Recombinant expression of S-layer proteins

ABSTRACT

The invention concerns a process for the recombinant production of S-layer proteins in gram-negative host cells. Furthermore the nucleotide sequence of a new S-layer gene and processes for the production of modified S-layer proteins are disclosed.

The present invention concerns processes for the recombinant productionof S-layer proteins and modified S-layer proteins in gram-negative hostcells.

Crystalline bacterial cell surface layers (S-layers) form the outermostcell wall component in many eubacteria and most of the archaebacteria(Sleytr et al. (1988), Crystalline Bacterial Cell Surface Layers,“Springer Verlag Berlin”; Messner and Sleytr, Adv. Microb. Physiol. 33(1992), 213-275). Most of the presently known S-layer proteins arecomposed of identical proteins or glycoproteins which have apparentmolecular weights in the range of 40,000 to 220,000. The components ofS-layers are self-assembling and most of the lattices have an oblique(p2), quadratic (p4) or hexagonal (p6) symmetry. The functions ofbacterial S-layers are still not completely understood but due to theirlocation on the cell surface the porous crystalline S-layers probablyserve mainly as protective coatings, molecular sieves or to promote celladhesion and surface recognition.

Genetic data and sequence information are known for various S-layergenes from microorganisms. A review may be found in Peyret et al., Mol.Microbiol. 9 (1993), 97-109. Explicit reference is made to these data.The sequence of the sbsA gene coding for the S-layer protein ofB.stearothermophilus PV72 and a process for cloning it are stated inKuen et al. (Gene 145 (1994), 115-120). B.stearothermophilus PV72 is agram-positive bacterium which is covered with a hexagonally arrangedS-layer. The main component of the S-layer is a 128 kd protein which isthe most frequent protein in the cell with a proportion of about 15%relative to the total protein components. Various strains ofB.stearothermophilus have been characterized which differ with regard tothe type of the S-layer lattice, the molecular weight and glycosilationof the S-layer components (Messner and Sleytr (1992), supra).

The German Patent Application P 44 25 527.6 discloses the signalpeptide-coding section of the S-layer gene from B.stearothermophilus andthe amino acid sequence derived therefrom. The cleavage site between thesignal peptide and the mature protein is located between position 30 and31 of the amino acid sequence. The signal peptide-coding nucleic acidcan be operatively linked to a protein-coding nucleic acid and can beused for the recombinant production of proteins in a process in which atransformed host cell is provided, the host cell is cultured underconditions which lead to an expression of the nucleic acid and toproduction and secretion of the polypeptide coded thereby and theresulting polypeptide is isolated from the culture medium. Prokaryoticorganisms are preferably used as host cells in particular gram-positiveorganisms of the genus bacillus.

Surprisingly it was found that the recombinant production of S-layerproteins is not only possible in gram-positive prokaryotic host cellsbut also in gram-negative prokaryotic host cells. In this case theS-layer protein is not formed in the interior of the host cell in theform of ordered inclusion bodies but rather unexpectedly in the form ofordered monomolecular layers.

Hence one subject matter of the present invention is a process for therecombinant production of S-layer proteins characterized in that (a) agram-negative prokaryotic host cell is provided which is transformedwith a nucleic acid coding for an S-layer protein selected from (i) anucleic acid which comprises the nucleotide sequence shown in SEQ ID NO.1 from position 1 to 3684 optionally without the section coding for thesignal peptide, (ii) a nucleic acid which comprises a nucleotidesequence corresponding to the nucleic acid from (i) within the scope ofthe degeneracy of the genetic code and (iii) a nucleic acid whichcomprises a nucleotide sequence which hybridizes with the nucleic acidsfrom (i) or/and (ii) under stringent conditions; (b) the host cell iscultured under conditions which lead to an expression of the nucleicacid and to production of the polypeptide coded thereby and (c) theresulting polypeptide is isolated from the host cell.

The term “stringent hybridization” is understood within the sense of thepresent invention to mean that a hybridization still also occurs afterwashing at 55° C., preferably 60° C. in an aqueous low salt buffer (e.g.0.2× SSC) (see also Sambrook et al. (1989), Molecular Cloning. ALaboratory Manual).

The process according to the invention is carried out in gram-negativeprokaryotic host cells. In this process an ordered S-layer proteinstructure is surprisingly obtained in the cell interior. Enterobacteria,in particular E. coli, are preferably used as host cells.

The E. coli strain pop2135 which was deposited on 31.01.1996 at the“Deutsche Sammlung von Mikroorganismen und Zelikulturen GmbH”,Mascheroder Weg 1b, D 38124 Braunschweig under the file number DSM 10509is particularly preferred

The process according to the invention can also be used to isolaterecombinant S-layer proteins. For this one uses a nucleic acid codingfor the S-layer protein which contains one or several insertions whichcode for peptide or polypeptide sequences. These insertions can, on theone hand, only code for peptides with a few amino acids e.g. 1-25 aminoacids. On the other hand, the insertions can also code for largerpolypeptides of for example up to 1000 amino acids and preferably up to500 amino acids without loss of the ability of the S-layer protein toform a correctly folded structure. In addition to the insertions therecombinant S-layer protein can also have amino acid substitutions, inparticular substitutions of individual amino acids in the region of theinsertion sites as well as optionally deletions of individual aminoacids or short amino acid sections of up to 30 amino acids.

Regions between the positions 1-1200 and 2200-3000 of the nucleotidesequence shown in SEQ ID NO.1 are preferred as insertion sites forpolypeptide-coding sequences. Particularly preferred insertion sites arethe NruI cleavage site at position 582, the PvuII cleavage site atposition 878, the SnaB-I cleavage site at position 917, the PvuIIcleavage site at position 2504 and the PvuII cleavage site at position2649. It was already possible to demonstrate the insertion of a nucleicacid coding for streptavidin into the NruI cleavage site at position581.

The peptide or polypeptide-coding insertions are preferably selectedfrom nucleotide sequences which code for cysteine residues, regions withseveral charged amino acids, e.g. Arg, Lys, Asp or Glu, or Tyr residues,DNA-binding epitopes, antigenic, allergenic or immunogenic epitopes,metal-binding epitopes, streptavidin, enzymes, cytokines orantibody-binding proteins.

A particularly preferred example of an insertion into the nucleic acidcoding for the S-layer protein is a nucleotide sequence coding forstreptavidin. In this manner it is possible to obtain universal carriermolecules which are suitable for coupling biotinylated reagents and fordetection in immunological or hybridization test procedures.

A further preferred example of insertions are antigenic, allergenic orimmunogenic epitopes e.g. epitopes from pathogenic microorganisms suchas bacteria, fungi, parasites etc. and viruses, or epitopes from plantsor epitopes against endogenous substances e.g. cytokines as well asagainst toxins in particular endotoxins. Particularly preferred examplesof immunogenic epitopes are epitopes from herpes viruses such as theherpes virus 6 or pseudorabies virus (Lomniczi et al., J. Virol. 49(1984), 970-979), in particular epitopes from the genes gB, gC or/andgD, or foot-and-mouth disease virus (FMDV), in particular epitopes fromthe gene sections which code for VP1, VP2 or/and VP3. The immunogenicepitopes can be selected such that they promote an antibody-mediatedimmune reaction or/and the production of a cellular immune reaction e.g.by stimulation of T cells. Examples of suitable allergenic epitopes arebirch pollen allergens e.g. Bet v I (Ebner et al., J. Immunol. 150(1993) 1047-1054). Antigenic epitopes are additionally particularlypreferred which are able to bind and filter out endogenous or exogenoussubstances such as cytokines or toxins from serum or other body fluids.Such epitopes can include components of cytokine or toxin receptors orof antibodies against cytokines or toxins.

On the other hand the insertions can also code for enzymes. Preferredexamples are enzymes for the synthesis of polyhydroxybutyric acid e.g.PHB synthase.

Incorporation of PHB synthase into the S-layer can lead to the formationof a molecular spinning nozzle under suitable conditions when thesubstrate hydroxybutyric acid is provided. A further preferred exampleof an enzyme is bacterial luciferase. In this case when the enzymesubstrate, an aldehyde, is supplied and 02 is present, a molecular lasercan be obtained.

Insertions are likewise preferred which code for cytokines such asinterleukins, interferones or tumour necrosis factors. These moleculescan for example be used in combination with immunogenic epitopes toprepare vaccines.

Finally insertions are also preferred which code for antibody bindingproteins such as protein A or protein G or for DNA-binding or/andmetal-binding epitopes such as the leucine zipper, zinc finger etc.

Thus for the first time a cell is provided by the present inventionwhich contains immobilized recombinant polypeptides in a native forme.g. active enzymes in the cytoplasm. In this manner 50,000-200,000 e.g.ca. 100,000 recombinant molecules can be immobilized per m² recombinantS-layer. Up to 3000 m² S-layer can be obtained per kg recombinant E.coli cells.

In the method according to the invention the nucleic acid coding for theS-layer protein is preferably used in operative linkage with a nucleicacid coding for a signal peptide of gram-positive bacteria i.e. thesignal peptide-coding nucleic acid is located on the 5′ side of theS-layer protein-coding nucleic acid. Surprisingly it was found that thepresence of such signal peptide sequences, which are not cleaved in thegram-negative host cells used in the invention, can improve thestability of the S-layer structures. The nucleic acid coding for thesignal peptide particularly preferably comprises (a) the signalpeptide-coding section of the nucleotide sequence shown in SEQ ID NO. 1,(b) a nucleotide sequence corresponding to the sequence from (a) withinthe scope of the degeneracy of the genetic code or/and (c) a nucleotidesequence which is at least 80% and in particular at least 90% homologousto the sequences from (a) or/and (b).

Yet a further subject matter of the present invention is a nucleic acidwhich codes for a recombinant S-layer protein and is selected from (i) anucleic acid which comprises the nucleotide sequence shown in SEQ IDNO.1 from position 1 to 3684 optionally without the signalpeptide-coding section (ii) a nucleic acid which comprises a nucleotidesequence corresponding to a nucleic acid from (i) within the scope ofthe degeneracy of the genetic code and (iii) a nucleic acid whichcomprises a nucleotide sequence which hybridizes under stringentconditions with the nucleic acids from (i) or/and (ii).

The coding nucleotide sequence of the S-layer gene sbsA fromB.stearothermophilus including the signal peptide-coding section isshown in SEQ ID NO. 1. The signal peptide-coding section extends fromposition 1 to 90 of the nucleotide sequence shown in SEQ ID NO. 1. Thesection coding for the mature SbsA polypeptide extends from position 91to 3684.

The sbsA gene of B.stearothermophilus codes for a protein with a totalof 1228 amino acids including an N-terminal signal peptide with 30 aminoacids (SEQ ID NO. 2). The cleavage site between the signal peptide andthe mature protein is located between position 30 and 31 of the aminoacid sequence. The signal peptide has a basic amino-terminal domainfollowed by a hydrophobic domain.

Sequence comparisons with other signal peptides indicate a certainhomology to signal peptides of extracellular proteins in bacilli such asalkaline phosphatase and neutral phosphatase of B.amyloliquefaciens(Vasantha et al., J. Bacteriol. 159 (1984), 811-819) as well as with thesignal peptides for the B.sphaericus gene 125 (Bowditch et al., J.Bacteriol. 171 (1989), 4178-4188) and the OWP qene of B.brevis (Tsuboiet al., J. Bacteriol. 168 (1986), 365-373).

A further subject matter of the present invention is a recombinantvector which contains at least one copy of a nucleic acid according tothe invention. The vector is preferably replicatable in prokaryotes. Thevector is particularly preferably a prokaryotic plasmid.

Yet a further subject matter of the present invention is a host cellwhich is transformed with a nucleic acid or a recombinant vectoraccording to the present invention. The cell is preferably agram-negative prokaryotic organism and most preferably an E. coli cell.The cell according to the invention can contain a recombinant S-layerstructure in its interior. Methods for the transformation of cells withnucleic acids are general state of the art (cf. Sambrook et al., supra)and therefore do not need to be elucidated.

Yet a further subject matter of the present invention is a recombinantS-layer protein which contains at least one peptide insertion or/andpolypeptide insertion within the amino acid sequence shown in SEQ ID NO.2. Preferred examples of peptide insertions and polypeptide insertionshave already been elucidated.

A recombinant S-layer structure can be assembled from recombinantS-layer protein molecules according to the invention which contain atleast one recombinant S-layer protein according to the invention as asubunit. Furthermore it is preferred that the S-layer structureaccording to the invention also contains non-modified S-layer proteinsas diluent molecules. The non-modified S-layer proteins are preferablypresent in a molar proportion of 10-99% relative to the total S-layerproteins.

The S-layer structure according to the invention can comprise severallayers that are covalently linked together or by means of affinitybinding. Covalent linkages can for example be introduced by insertionsof cysteine residues and a subsequent formation of cystine bridges.Linkages by affinity binding comprise for example antibody-antigen,antibody-protein A or antibody-protein G or streptavidin-biotininteractions.

S-layer structures which contain recombinant S-layer proteins canoptionally also be prepared in a carrier-bound form. For this theS-layer structure can be reassembled from individual units in thepresence of a peptidoglycan carrier to for example producepeptido-glycan layers which are coverged on one or on both sides with anS-layer structure. Another method of preparing carrier-bound S-layerstructures is to produce an S-layer layer at an interface between twomedia e.g. water/air and to immobilize this layer on a solid phase e.g.a filter membrane (cf. e.g. Pum and Sleytr (1994), Thin Solid Films 244,882-886; Kupcu et al., (1995), Biochim. Biophys. Acta 1235, 263-269).

The recombinant S-layer proteins and S-layer structures according to theinvention are suitable for a multitude of applications. An applicationas a vaccine or adjuvant is particularly preferred in which caserecombinant S-layer proteins are used which contain immunogenic epitopesof pathogens and/or endogenous immuno-stimulatory polypeptides such ascytokines. In this application it is not absolutely necessary to purifythe recombinant S-layer proteins. Instead they can for example be usedin combination with a bacterial ghost which optionally containsadditional immunogenic epitopes in its membrane.

The preparation of suitable “bacterial ghosts” is described for examplein the International Patent application PCT/EP91/00967 to whichreference is herewith made. In this application modified bacteria aredisclosed which are obtainable by transformation of a gram-negativebacterium with the gene of a lytically active membrane protein frombacteriophages, with the gene of a lytically active toxin releaseprotein or with genes which contain partial sequences thereof which codefor lytic proteins, culturing the bacterium, expression of this lysisgene and isolation of the resulting bacterial ghost from the culturemedium.

A recombinant protein, which is obtainable by expression of arecombinant DNA in these gram-negative bacteria, can be bound to themembrane of these bacteria as described in the European Patent 0 516655. This recombinant DNA comprises a first DNA sequence which codes fora hydrophobic, non-lytically active membrane-integrating protein domainwhich has an a-helical structure and is composed of 14-20 amino acidswhich can be flanked N- and C-terminally by 2-30 arbitrary amino acidsin each case. A second DNA sequence is in operative linkage with thisfirst DNA sequence which codes for a desired recombinant protein.Furthermore the gram-negative bacterium contains a third DNA sequencewhich is under a different control from the first and second DNAsequences and codes for a lytically active membrane protein frombacteriophages or a lytically active toxin release protein or for theirlytically active components. So-called “bacterial ghosts” are obtainedby expression and lysis of such recombinant gram-negative bacteria whichcontain an intact surface structure with immunogenic epitopes bound tothe surface.

When these bacterial ghosts are combined with recombinant S-layersaccording to the invention vaccines and adjuvants can be produced whichhave particularly advantageous properties.

A further particularly preferred application for recombinant S-layerproteins and S-layer structures is their use as an enzyme reactor. Suchan enzyme reactor can for example be formed by a cell which contains arecombinant S-layer structure according to the invention in itsinterior. On the other hand the enzyme reactor can also be formed fromisolated and in vitro reassembled S-layer structures or combinations ofvarious S-layer structures.

It was found that the gram-positive bacterium B.stearothermophilus PV72has an additional S-layer protein in addition to SbsA which issubsequently denoted as SbsB (Sara and Sleytr (1994), J. Bacteriol. 176,7182-7189). It was possible to isolate and characterize the sbsB gene byamplification using suitable nucleic acid primers. The coding nucleotidesequence of the S-layer gene sbsB from B.stearothermophilus includingthe signal peptide-coding section which extends from position 1 to 93 ofthe nucleic acid sequence is shown in SEQ ID NO.5. The amino acidsequence derived therefrom is shown in SEQ ID NO.6. The sbsB gene codesfor a protein with a total of 921 amino acids including an N-terminalsignal peptide with 31 amino acids.

One subject matter of the present invention is hence a nucleic acidwhich codes for an S-layer protein and is selected from

(i) a nucleic acid which comprises the nucleotide sequence from position1 to 2763 shown in SEQ ID NO.5 optionally without the signalpeptide-coding section,

(ii) a nucleic acid which comprises a nucleotide sequence correspondingto the nucleic acid from (i) within the scope of the degeneracy of thegenetic code and

(iii) a nucleic acid which comprises a nucleotide sequence thathybridizes with the nucleic acids from (i) or/and (ii) under stringentconditions.

As in the case of the sbsA gene, it is also possible to insert at leastone nucleic acid insertion coding for a peptide or polypeptide into thesbsB gene within the region coding for the S-layer protein. With regardto preferred examples of insertions in the sbsB gene reference is madeto the previous statements regarding the sbsA gene.

Yet a further subject matter of the present invention is a vector whichcontains at least one copy of an sbsB gene optionally containing aninsertion. This vector can be replicated in eukaryotes, prokaryotes orin eukaryotes and prokaryotes. It can be a vector that can be integratedinto the genome of the host cell or a vector which is presentextrachromosomally. The vector according to the invention is preferablya plasmid in particular a prokaryotic plasmid.

Yet a further subject matter of the present invention is a host cellwhich is transformed with an sbsB gene wherein the sbsB gene optionallycan contain an insertion. The host cell can be a eukaryotic as well as aprokaryotic cell. The cell is preferably a prokaryotic organism.Gram-positive organisms e.g. organisms of the genus bacillus as well asgram-negative organisms such as enterobacteria in particular E. coli arepreferred. Methods for transforming eukaryotic and prokaryotic cellswith nucleic acids are known and therefore do not need to be elucidatedin detail.

The present invention also concerns an SbsB protein i.e. an S-layerprotein which is coded by a nucleic acid as defined above. RecombinantSbsB proteins are particularly preferred which contain one or severalpeptide or/and polypeptide insertions within the sbsB sequence. The SbsBpart of a polypeptide according to the invention particularly preferablyhas a homology of at least 80% and in particular of at least 90% to theamino acid sequence shown in SEQ ID NO.6.

A recombinant S-layer structure can also be assembled from therecombinant SbsB-S-layer protein molecules analogous to the recombinantSbsA-S-layer structure. In this structure the non-modified S-layerproteins are preferably present in a molar proportion of 10-99% relativeto the total S-layer proteins.

The applications for the recombinant SbsB-S-layer proteins and S-layerstructures according to the invention also correspond to theapplications for SbsA mentioned above. In this connection its use as avaccine or adjuvant or as an enzyme reactor is noteworthy.

Recombinant S-layer proteins are obtainable by a process in which

(a) a host cell is provided which contains a nucleic acid coding for anS-layer protein which contains a peptide-coding or polypeptide-codinginsertion within the region coding for the S-layer protein,

(b) the host cell is cultured under conditions which lead to anexpression of the nucleic acid and to production of the polypeptidecoded by it and

(c) the resulting polypeptide is isolated from the host cell or from theculture medium.

In a first preferred embodiment of this process a recombinantSbsA-S-layer protein is prepared i.e. the nucleic acid coding for therecombinant S-layer protein is selected from

(i) a nucleic acid which comprises the nucleotide sequence from position1 to 3684 shown in SEQ ID NO.1 optionally without the signalpeptide-coding section,

(ii) a nucleic acid which comprises a nucleotide sequence correspondingto the nucleic acid from (i) within the scope of the degeneracy of thegenetic code and

(iii) a nucleic acid which comprises a nucleotide sequence whichhybridizes with the nucleic acids from (i) or/and (ii) under stringentconditions.

In a second preferred embodiment a recombinant SbsB-S-layer protein isprepared i.e. the nucleic acid coding for the recombinant S-layerprotein is selected from

(i) a nucleic acid which comprises the nucleotide sequence from position1 to 2763 shown in SEQ ID NO.5 optionally without the signalpeptide-coding section,

(ii) a nucleic acid which comprises a nucleotide sequence correspondingto the nucleic acid from (i) within the scope of the degeneracy of thegenetic code and

(iii) a nucleic acid which comprises a nucleotide sequence whichhybridizes with the nucleic acids from (i) or/and (ii) under stringentconditions.

In addition to the recombinant SbsA and SbsB-S-layer proteins fromB.stearothermophilus it is, however, also possible to preparerecombinant S-layer proteins from other organisms (cf. e.g. Peyret etal., (1993), supra).

The recombinant S-layer proteins can on the one hand be produced in aheterologous host cell i.e. in a host cell which originally contains noS-layer gene. Examples of such heterologous host cells are gram-negativeprokaryotic organisms such as E. coli.

However, the heterologous expression of S-layer proteins can also takeplace in gram-positive prokaryotic organisms such as B. subtilis. Forthis integration vectors are preferably used which contain a nativeor/and a recombinant S-layer gene. When the native signal sequences areused the S-layer proteins are secreted into the culture supernatant.

However, it is often preferable to produce the recombinant S-layerproteins in homologous host cells i.e. host cells which originallycontain a natural S-layer gene. In one embodiment of this homologousexpression the recombinant S-layer gene is introduced into the host cellin such a way that the host cell is still able to express a furtherS-layer gene which codes for a non-modified S-layer protein. Thenon-modified S-layer protein is preferably capable of forming an S-layerstructure that is compatible with the recombinant S-layer protein. Anexample of this embodiment of homologous expression is aB.stearothermophilus PV72 cell which contains intact natural sbsA genesor/and sbsB genes and is transformed with a plasmid which contains arecombinant S-layer gene.

In a second embodiment the homologous expression can occur in a hostcell in which the intact S-layer gene originally present has beeninactivated. Consequently in this embodiment no further S-layer gene isexpressed in the host cell which codes for a non-modified S-layerprotein which is able to form a compatible S-layer structure with therecombinant S-layer protein. A specific example of such a host cell is aB.stearothermophilus PV72 cell in the genome of which a gene coding fora recombinant S-layer protein has been introduced, e.g. by homologousrecombination, which replaces the original S-layer gene. A furtherexample of such a host cell is a B.stearothermophilus cell in which thenative S-layer gene has been inactivated e.g. by site-specificmutagenesis or/and homologous recombination and is transformed with avector containing a recombinant S-layer gene.

Gram-positive prokaryotic organisms are usually used as host cells forthe homologous expression of recombinant S-layer genes.B.stearothermophilus PV72 is particularly preferred as a host cell whichcan be cultured at a high temperature in a defined synthetic medium(Schuster et al., (1995), Biotechnol. and Bioeng. 48: 66-77).

The present invention is further elucidated by the following examplesand figures.

SEQ ID NO.1 shows the complete nucleotide sequence of the coding sectionof the S-layer gene sbsA of B.stearothermophilus;

SEQ ID NO.2 shows the amino acid sequence derived therefrom;

SEQ ID NO.3 shows the nucleotide sequence of the primer T5-X;

SEQ ID NO.4 shows the nucleotide sequence of the primer E;

SEQ ID NO.5 shows the complete nucleotide sequence of the coding sectionof the S-layer gene sbsB of B.stearothermophilus;

SEQ ID NO.6 shows the amino acid sequence derived therefrom;

SEQ ID NO.7 shows the nucleotide sequence of a partial fragment of thestreptavidin gene;

SEQ ID NO.8 shows the nucleotide sequence of the primer NIS 2AG;

SEQ ID NO.9 shows the nucleotide sequence of the primer LIS C3;

FIG. 1 shows a schematic representation of the sbsA PCR fragment used toprepare the recombinant vector pBK4;

FIG. 2 shows a schematic representation of peptide insertions in theamino acid sequence of the SbsA S-layer protein and

FIG. 3 shows a schematic representation of amino acid substitutions andamino acid insertions in recombinant S-layer proteins.

EXAMPLES

1. Bacterial Strains, Media and Plasmids

Gram-positive bacteria of the strain Bacillus stearo-thermophilus PV72were cultured at 58° C. in SVIII medium (Bartelmus and Perschak, Z.Zuckerrind. 7 (1957), 276-281). Bacteria of the strain E. coli pop2135(endA, thi, hsdR, malT, cI857, XpR, malPQ) were cultured in LB medium(Sambrook et al., (1989), supra). Ampicillin was added to the medium ata final concentration of 100 μg/ml to select for transformants. Theplasmid pPLcAT10 (kpL, bla, colEl) (Stanssens et al., Gene 36 (1985),211-223) was used as the cloning vector.

2. Manipulation of DNA Fragments

Restriction analysis of DNA, agarose gel electrophoresis and cloning ofDNA fragments were carried out according to the standard methodsdescribed in Sambrook et al. (1989), supra.

Competent cells were transformed by electroporation using a Bio-Rad genepulser (Bio-Rad Laboratories, Richmond, Calif. USA) according to themanufacturer's instructions.

Plasmid DNA was isolated by the method of Birnboim and Doly (NucleicAcids Res. 7 (1979), 1513-1523). Chromosomal DNA was isolated accordingto the method described in Ausubel et al. (Current Protocols inMolecular Biology (1987), N.Y., John Wiley).

Restriction endonucleases and other enzymes were obtained fromBoehringer Mannheim, New England Biolabs or Stratagene and usedaccording to the manufacturer's instructions.

3. DNA Sequencing

The DNA sequences of the 5′ regions and the 3′ regions (including theregion coding for the signal sequence) of the gene sbsA in the vectorpPLcAT10 were determined by the dideoxy chain termination method ofSanger et al. The primers used for sequencing were constructed on thebasis of the already published sbsA sequence (Kuen et al. Gene 145(1994), 115-120).

4. PCR Amplification of sbsA

The PCR amplification of the sbsA gene was carried out in a reactionvolume of 100 μl in which 200 μM deoxynucleotides, 1 U Pfu-polymerase(Stratagene), 1× Pfu-reaction buffer, 0.5 μM of each oligonucleotideprimer and 100 ng genomic DNA from B.stearothermophilus as a templatewere present. The amplification was carried out for 30 cycles in athermocycler (Biomed thermocycler 60). Each cycle was composed of adenaturing step of 1.5 min at 95° C., an annealing step of 1 min at 56°C. and 1 min at 50° C. as well as an extension step of 2 min at 72° C.

The primer T5-X shown in the sequence protocol as SEQ ID NO.3 whichflanks the 5′ region of sbsA and contains an XbaI site and the primer Eshown in the sequence protocol in SEQ ID NO.4 which flanks the 20nucleotide upstream region of the transcription terminator of the sbsAsequence and contains a BamHI site were used as primers.

The products amplified by PCR were electrophoretically separated on a0.8% agarose gel and purified for cloning using the system from GeneClean (BI0101 La Jolla, Calif. USA).

5. Cloning of the sbsA Gene into the Vector pPLcAT10

The sbsA gene obtained by PCR with a length of 3.79 kb was purified andcleaved with the restriction endonucleases XbaI and BamHI. The resultingXbaI-BamHI fragment was cloned into the corresponding restriction sitesof the vector pPLcAT10 so that the sbsA gene was under transcriptionalcontrol of the pL promoter located upstream. The ATG start codon of thesbsA sequence was reconstructed by the cloning procedure. The clonedsbsA sequence contained the N-terminal signal sequence of sbsA and ended20 nt after the transcription terminator. After ligation of the vectorDNA with the sbsA fragment, the E. coli strain pop2135 was transformedby electro-transformation. The resulting clones were subjected to a DNArestriction analysis. A positive clone was sequenced in order to verifythe correct sequence transitions at the 5′ and 3′ ends. This clone wasnamed pBK4.

A schematic representation of the 3.79 kb XbaI sbsA fragment and itslocation in the multiple cloning site of the plasmid pBK4 is shown inFIG. 1 (abbreviations: tT: transcription terminator; ori: origin of theDNA replication; amp: ampicillin resistance gene).

6. Recombinant Expression of the sbsA Gene in E. coli

E. coli pop2135/pBK4 cells were cultured at 28° C. until an opticaldensity OD₆₀₀ of 0.3 was reached. Then the expression of sbsA wasinduced by increasing the culture temperature from 28° C. to 42° C. 1.5ml aliquots were taken before and 1, 2, 3 and 5 hours after induction ofthe sbsA expression. E. coli pop2135/pPLcAT10 (cultured under the sameconditions) and B.stearothermophilus PV72 were used as controls.

Culture supernatants and cell extracts from all samples were examinedfor the expression of S-layer proteins by SDS-PAGE and Westernimmunoblotting.

An additional strong protein band with the same molecular weight as thewild type SbsA protein was found in extracts from E. coli cellstransformed with pBK4. No degradation products of SbsA itself were foundin a period of up to 5 hours after induction of expression. Thuspresumably the S-layer protein sbsA is stable in E. coli and is notdegraded by proteases.

A densitometric determination of the relative amount of SbsA protein wascarried out. At a time point of 4 hours after induction the sbsA proteinwas in a proportion of ca. 16% relative to the total cellular protein.

The SbsA protein produced in E. coli migrated in the SDS gel slightlymore slowly than the natural SbsA protein from B.stearothermophilus.Experiments to determine the N-terminal amino acid sequence of the SbsAprotein by Edman degradation were not successful due to a blocking ofthe N-terminus. Thus presumably the signal sequence was not cleaved inE. coli.

A Western blot analysis of total cell extracts and culture supernatantsof E. coli/pBK4 also only yielded a single sbsA-specific protein bandwith a slightly higher molecular weight than wild type SbsA protein fromstearothermophilus.

For the Western blot the proteins were transferred onto a nitrocellulosemembrane and incubated with a polyclonal antiserum against SbsA fromrabbits. The preparation of this antiserum is described in Egelseer etal. (J. Bacteriol. 177 (1995), 1444-1451). A conjugate of goatanti-rabbit IgG and alkaline phosphatase was used to detect boundSbsA-specific antibodies.

No SbsA protein could be detected from supernatants from E. coli cellstransformed with pBK4 even after induction of sbsA gene expression. Thisshows that SbsA is not exported into the surrounding medium.

7. Location and Organisation of the S-Layer Protein SbsA in theCytoplasm of E. coli

Cells of E. coli pop2135/pBK4 which were harvested from cultures 1, 2, 3and 5 hours after induction of the S-layer protein expression wereexamined for the intra-cellular organisation of sbsA. Non-induced cellscultured at 28° C. and cells of B.stearothermophilus PV72 were examinedas controls.

For this whole cells of both organisms were fixed and embedded indetection resin according to the method of Messner et al. (Int.J.Syst.Bacteriol. 34 (1984), 202-210). Subsequently ultrathin sectionsof the embedded preparations were prepared and stained with uranylacetate.

The cytoplasm of non-induced E. coli cells exhibited the typicalgranular structure which did not change even when the OD of thesuspensions increased. Longitudinal sections of E. coli cells which wereharvested 1 hour after induction of the S-layer protein expressionexhibited parallel, leaf-like structures in the cytoplasm. From crosssections it was apparent that these structures have a concentricarrangement.

The amount of leaf-like structures considerably increased between 1 and2 hours after induction of the sbsA expression and afterwards remainedessentially constant.

The sbsA protein recombinantly produced in E. coli could also bedetected by immunogold labelling with sbsA-specific antibodies. Anordered structure of the recombinantly produced SbsA protein was alsofound with this detection method.

It was clearly apparent from these morphological data that the SbsAprotein did not aggregate to form irregular inclusion bodies but ratherformed monomolecular S-layer crystals. A remarkable property of theSbsA-S-layer layers assembled in E. coli was the concentric arrangementat defined distances. The presence of the signal sequence did notinterfere with correct assembly.

8. Preparation of Recombinant sbsA-S-Layer Genes

8.1 Insertion of a 6 bp Long DNA Sequence

A modified kanamycin cassette (1.3 kb) was used for the site-specificinsertion mutagenesis of the sbsA gene which was isolated by cleavage ofthe plasmid pWJC3 (obtained from W. T. McAllister, N.Y.) by SmaI. Thecassette was ligated into five different blunt-ended restriction sitesof the sbsA gene, i.e. into the NruI site at position bp 582 (pSL582),into the SnaBI site at position bp 917 (pSL917) and into each of thePvuII sites at positions bp 878 (pSL878), bp 2504 (pSL2504) and bp 2649(pSL2649). After selection of kanamycin-resistant clones, the cassettewas removed from the insertion site by cleavage with ApaI followed by areligation of the S-layer plasmid pBK4. The cutting out and religationprocedure left an insertion of 6 bp CCCGGG (ApaI restriction site). Thesystem of this linker insertion is shown schematically in FIG. 2.

The resulting recombinant S-layer genes code for modified sbsA proteinselongated by 2 amino acids.

The specific changes in the primary structure of the sbsA proteins areshown in FIG. 3. In the clone pSL582 the insertion led to theincorporation of glycine and proline between the amino acids 194 and 195at the N-terminus of the SbsA protein. The amino acids alanine andarginine were inserted in the clone pSL917 between the amino acids 306and 307. In the clone pSL2649 glycine and proline were inserted betweenthe amino acids at positions 883 and 884. An insertion of alanine andproline between the amino acids 293 and 294 was obtained in the clonepSL878. Furthermore the alanine at position 293 was substituted byglycine. In the clone pSL2504 the amino acids alanine and proline wereinserted between the amino acids 835 and 836 and the alanine at position835 was replaced by glycine.

All clones obtained by insertion mutagenesis retained their ability tosynthesise the S-layer protein.

In order to test the ability of the modified proteins to assemble intoS-layer structures, ultrathin longitudinal sections of whole cells whichhad been cultured for 4 hours under inductive conditions were preparedaccording to the procedure described in section 7. It was found that thecytoplasm of all five clones is filled with parallel, leaf-likestructures which follow the curve of the cell poles. There were nomorphological differences of the cytoplasm in the 5 different clonesexamined. Exactly the same leaf-like structures were found as in theassembly of the wild type SbsA protein in E. coli (section 7).

8.2 Insertion of a DNA Sequence Coding for Streptavidin

In order to examine whether the insertion of larger protein sequencesinto the SbsA protein can also be tolerated, a DNA fragment coding for apart of streptavidin (160 amino acids) provided with ApaI linkers (SEQID NO.7) was gene inserted into the ApaI restriction site of the sbsAclones pSL582, pSL878, pSL917 and pSL2649 prepared in the example onpage 1. The streptavidin sequence was inserted in SL582 in the codon197, in pSL878 between codon 295 and 296, in pSL917 in the codon 308 and309 and in pSL2649 in the codon 886. It was possible to detect theexpression of SbsA-streptavidin fusion proteins in all constructs bySDS-PAGE and immunoblots. It was found by EM analysis that a selfassembly of the S-layer structure was possible in the fusion proteinscontaining insertions in the codon 197 and between the codons 295 and296.

The SbsA-streptavidin fusion proteins can be isolated as monomers andreassembled to form homogeneous SbsA-streptavidin S-layers or mixedSbsA-streptavidin/SbsA-S-layers. They can be used to bind biotinylatedsubstances as well as to determine the binding capacity of enzymes andother bound molecules.

8.3 Insertion of a DNA Sequence Coding for BetvI

A DNA sequence coding for the open reading frame of BetvI (161 aminoacids) the main pollen allergen of the birch (Ferreira et al., J. Biol.Chem. 268 (1993), 19574-19580) was inserted at the ApaI site into thesbsA clone pSL878. It was possible to detect the expression of anSbsA-BetvI fusion protein which contained an immunologically activeBetvI domain.

The resulting fusion protein can be used for therapeutic or diagnosticpurposes. Hence it can be attempted by administration of the fusionprotein to convert a TH²-directed IgE antibody reaction into aTH1-mediated reaction against BetvI. In this manner it is possible tosuppress the occurrence of symptoms of a pollen allergy. FurthermoreSbsA-BetvI fusion proteins can be used to test for anti-BetvI antibodyconcentrations or/and to reduce high concentrations of anti-BetvI IgE.

8.4 Insertion of a DNA Sequence Coding for a Pseudorabies Virus Antigen

The DNA sequence coding for the gB epitope SmaBB (255 amino acids)(nucleotides 489-1224 corresponding to the coordinates according to theEMBL-Seq: HEHSSGP2) from the pseudorabies virus was inserted into SSpIsite of the sbsA gene after nt 3484 (between codon 1161 and 1162). Itwas possible to detect the expression of SbsA-SmaBB fusion proteins.

The fusion proteins can be used to test gB-specific immune reactions. AWestern blot analysis using a monoclonal antibody which corresponds tothe inserted sequence showed the immunological activity of the viraldomain within the recombinant SbsA-SmaBB proteins.

8.5 Insertion of a DNA Sequence Coding for the PHB Synthase (PhbC) fromAlcaligenes eutrophus H16

A regular arrangement of polypeptide structures with enzymatic activityon the surface of S-layers is an important goal in the production ofimmobilized enzymes within a living cell and in the case of the 590amino acid long PHB synthase for the production of a molecular machinefor biopolymer synthesis.

The phbc gene was isolated by PCR from the plasmid p4A (Janes et al.,Molecular characterisation of the poly-β-hydroxy-butyrate biosynthesisin Alcaligenes eutrophus H16. In: Novel Biodegradable Microbial Polymers(publisher Daves, E. A.), pp 175-190 (1990), Kluver, Dordrecht) as a1770 nt long DNA fragment (corresponding to an open reading frame of 590amino acids) and inserted into the ApaI cleavage site of the sbsA clonepSL878 to obtain the plasmid pSbsA-PhbC. It was possible to detect theexpression of an SbsA-PhbC fusion protein of ca. 195 kD in an E. colicell transformed with this plasmid. When two copies of the phbc genewere inserted one behind the other into the ApaI site of pSL878, it waspossible to detect the expression of a fusion protein of ca. 260 kD.

For a functional test of the enzymatic activity of the SbsA-PhbCconstruct, the E. coli cells which contained the plasmid pSbsA-PhbC wereco-transformed with the plasmid pUMS which contains the β-ketothiolase(PhbA) and the acetoacetyl-CoA reductase (PhbB) from A. eutrophus(Kalousek et al., Genetic engineering of PHB-synthase from Alcaligeneseutrophus H16. In: Proceedings of the International Symposium onBacterial Polyhydroxy-alkanoates, pp 426-427 (1993), publisher SchlegelH. G., Steinbuchel A. Goltze Press, Gbttingen). Thepoly-β-hydroxybutyrate formation in the co-transformed E. coli cells wasdetectable by staining with Sudan black, gas chromatography and electronmicroscopy. These findings show that the SbsA-PhbC construct isenzymatically active and represents a successful example of theimmobilization of enzymes on intracellular S-layer matrices.

8.6 Insertion of a DNA Sequence Coding for a Bacterial Luciferase Gene

A monocistronic LuxAB gene with a length of 2,070 nt which contains thefusion protein LuxAB composed of the two subunits LuxB and LuxB of thebacterial luciferase from Vibrio harveyi was isolated from the plasmidpT7-mut3 (Boylan et al., J. Biol. Chem. 264 (1989), 1915-1918) by PCRand inserted into the ApaI site of the clone pSL878 prepared in example8.1 to obtain the plasmid pBK878-LuxAB. It was possible to detect theexpression of an SbsA-PhbC fusion protein of ca. 207 kD in an E. colicell transformed with this plasmid. The enzymatic activity of the fusionprotein was demonstrated by the method described in Boylan et al.,Supra.

9. Isolation and Characterization of the sbsB Gene

The basis for the isolation of the sbsB gene was the amino acid sequenceof the N-terminus as well as the sequence of three internal peptides ofthe SbsB protein. Starting with these peptide sequences, degenerateoligonucleotide primers were constructed and used for the PCR. In thismanner a 1076 bp long PCR fragment from the chromosomal DNA ofB.stearothermophilus was amplified, cloned and sequenced (correspondingto position 100-1176 of the sequence shown in SEQ ID NO.5).

The method of inverse PCR was used to amplify the sections on the 5′side and 3′ side of the sbsB gene and stepwise overlapping DNA fragmentswere obtained with the aid of various primer combinations and sequenced.

The primer NIS 2AG shown in the sequence protocol as SEQ ID NO.8 whichcontains the 5′ region of sbsB as well as the primer LIS C3 shown in thesequence protocol of SEQ ID NO.9 which contains the 3′ region of sbsBwere used as primers to amplify the complete sbsB gene.

The PCR fragment obtained in this manner which contains the nucleotidesequence shown in SEQ ID NO.5 with 5′ and 3′ BamHI restriction cleavagesites was cloned as described in example 5 into the vector pPLcAT10 inwhich the expression takes place under the control of the lambda PLpromoter.

Furthermore the sbsB-PCR fragment with the 5′ side EcoRI and 3′ sideBamHi cleavage site were cloned into the vector pUC18 in which theexpression took place under the control of the lac promoter.

The detection of the sbsB expression was carried out as described inexamples 6 and 7 by SDS gel electrophoresis and electron microscopy.

10. Preparation of Recombinant sbsB-S-Layer Genes

Recombinant sbsB genes were prepared analogously to the methodsdescribed in example 8.

Thus in accordance with the method described in example 8.1, a 6 nt longDNA sequence containing an ApaI restriction cleavage site was introducedat various positions into the sbsB-layer gene. The recombinant sbsBclones pAK407, pAK481 and pAK1582 with ApaI cleavage sites at nt 407(codon 136), 481 (codon 161/162) and 1582 (codon 528/529) were obtainedin this manner. These clones obtained by insertion mutagenesis retainedtheir ability to synthesize the S-layer protein and form S-layerstructures.

Analogously to the method described in example 8.2, a DNA fragmentcoding for streptavidin was inserted into the ApaI restriction sites ofthe sbsB clones pAK407 and pAK481.

Analogously to example 8.4, a DNA sequence coding for the gB epitopeSmaBB was inserted into the ApaI cleavage sites of the sbsB clonespAK481 and pAK1582. It was possible to detect the expression ofsbsB-SmaB fusion proteins of ca. 130 kD in the E. coli cells transformedwith the resulting recombinant plasmids. When two copies of the SmaBBepitopes were inserted one behind the other into the ApaI cleavage siteof pAK481 it was possible to detect the expression of a fusion proteinof ca. 157 kD. The SmaBB domains of the fusion proteins were recognizedby specific antibodies.

Analogously to example 8.6 it was possible to detect the expression of a175 kD SbsB-LuxAB fusion protein when the LuxAB sequence was insertedinto the ApaI cleavage site of pAK407.

11. Heterologous Expression of sbsA and sbsB in Bacillus subtilis

The integration vector pX (Kim, L., Mogk, A. and Schumann W., Gene 181(1996), 71-76: A xylose-inducible Bacillus subtilis integration vectorand its application) was used for the heterologous expression of sbsAand sbsB in B. subtilis. The S-layer genes in the resulting recombinantexpression vectors are under the transcriptional control of the xylpromoter.

Transformants of B.subtilis containing an S-layer gene integrated in thechromosome exhibited an expression of large amounts of S-layer proteinsin the supernatant of the cells which was inducible by addition ofxylose to the growth medium. This shows that the signal sequences ofsbsA and sbsB are recognized by the B. subtilis cell.

In an analogous manner it was possible to achieve a heterologousexpression of recombinant sbsA and sbsB layer genes in B. subtilis.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS:  10 <210> SEQ ID NO 1 <211> LENGTH: 3687<212> TYPE: DNA <213> ORGANISM: Bacillus stearothermophilus<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(3684)<221> NAME/KEY: sig_peptide <222> LOCATION: (1)..(90)<221> NAME/KEY: mat_peptide <222> LOCATION: (91)..(3684)<400> SEQUENCE: 1 atg gat agg aaa aaa gct gtg aaa cta gca ac#a gca agt gct att gca       48Met Asp Arg Lys Lys Ala Val Lys Leu Ala Th #r Ala Ser Ala Ile Ala-30                 - #25                 - #20                 - #15gca agt gca ttt gtc gct gca aat cca aac gc#t tct gaa gcg gct aca       96Ala Ser Ala Phe Val Ala Ala Asn Pro Asn Al #a Ser Glu Ala Ala Thr                -10   #                -5   #            -1   1gat gta gca aca gta gta agc caa gca aaa gc#a cag ttc aaa aaa gca      144Asp Val Ala Thr Val Val Ser Gln Ala Lys Al #a Gln Phe Lys Lys Ala          5         #          10         #          15tac tat act tac agc cat aca gta acg gaa ac#t ggt gaa ttc cca aac      192Tyr Tyr Thr Tyr Ser His Thr Val Thr Glu Th #r Gly Glu Phe Pro Asn     20              #     25              #     30att aac gat gta tat gct gaa tac aac aaa gc#g aaa aaa cga tac cgt      240Ile Asn Asp Val Tyr Ala Glu Tyr Asn Lys Al #a Lys Lys Arg Tyr Arg 35                  # 40                  # 45                  # 50gat gcg gta gca tta gtg aat aaa gca ggt gg#c gcg aaa aaa gac gct      288Asp Ala Val Ala Leu Val Asn Lys Ala Gly Gl #y Ala Lys Lys Asp Ala                 55  #                 60  #                 65tac tta gct gat tta caa aaa gaa tat gaa ac#t tac gtt ttc aaa gca      336Tyr Leu Ala Asp Leu Gln Lys Glu Tyr Glu Th #r Tyr Val Phe Lys Ala             70      #             75      #             80aac cct aaa tct ggc gaa gct cgt gta gca ac#t tac atc gat gct tac      384Asn Pro Lys Ser Gly Glu Ala Arg Val Ala Th #r Tyr Ile Asp Ala Tyr         85          #         90          #         95aac tat gca aca aaa tta gac gaa atg cgc ca#a gag cta gag gct gct      432Asn Tyr Ala Thr Lys Leu Asp Glu Met Arg Gl #n Glu Leu Glu Ala Ala    100               #   105               #   110gtt caa gca aaa gat tta gaa aaa gca gaa ca#a tac tat cac aaa att      480Val Gln Ala Lys Asp Leu Glu Lys Ala Glu Gl #n Tyr Tyr His Lys Ile115                 1 #20                 1 #25                 1 #30cct tat gaa att aaa act cgc aca gtc att tt#a gat cgc gta tat ggt      528Pro Tyr Glu Ile Lys Thr Arg Thr Val Ile Le #u Asp Arg Val Tyr Gly                135   #               140   #               145aaa aca act cgt gat tta ctt cgc tct aca tt#t aaa gca aaa gca caa      576Lys Thr Thr Arg Asp Leu Leu Arg Ser Thr Ph #e Lys Ala Lys Ala Gln            150       #           155       #           160gaa ctt cgc gac agc tta att tat gat att ac#c gtt gca atg aaa gcg      624Glu Leu Arg Asp Ser Leu Ile Tyr Asp Ile Th #r Val Ala Met Lys Ala        165           #       170           #       175cgc gaa gta caa gac gct gtg aaa gca ggc aa#t tta gac aaa gct aaa      672Arg Glu Val Gln Asp Ala Val Lys Ala Gly As #n Leu Asp Lys Ala Lys    180               #   185               #   190gct gct gtt gat caa atc aat caa tac tta cc#a aaa gta aca gat gct      720Ala Ala Val Asp Gln Ile Asn Gln Tyr Leu Pr #o Lys Val Thr Asp Ala195                 2 #00                 2 #05                 2 #10ttc aaa act gaa cta aca gaa gta gcg aaa aa#a gca tta gat gca gat      768Phe Lys Thr Glu Leu Thr Glu Val Ala Lys Ly #s Ala Leu Asp Ala Asp                215   #               220   #               225gaa gct gcg ctt act cca aaa gtt gaa agt gt#a agt gcg att aac act      816Glu Ala Ala Leu Thr Pro Lys Val Glu Ser Va #l Ser Ala Ile Asn Thr            230       #           235       #           240caa aac aaa gct gtt gaa tta aca gca gta cc#a gtg aac gga aca cta      864Gln Asn Lys Ala Val Glu Leu Thr Ala Val Pr #o Val Asn Gly Thr Leu        245           #       250           #       255aaa tta caa ctt tca gct gct gca aat gaa ga#t aca gta aac gta aat      912Lys Leu Gln Leu Ser Ala Ala Ala Asn Glu As #p Thr Val Asn Val Asn    260               #   265               #   270act gta cgt atc tat aaa gtg gac ggt aac at#t cca ttt gcc ctt aat      960Thr Val Arg Ile Tyr Lys Val Asp Gly Asn Il #e Pro Phe Ala Leu Asn275                 2 #80                 2 #85                 2 #90acg gca gat gtt tct tta tct aca gac gga aa#a act atc act gtg gat     1008Thr Ala Asp Val Ser Leu Ser Thr Asp Gly Ly #s Thr Ile Thr Val Asp                295   #               300   #               305gct tca act cca ttc gaa aat aat acg gag ta#t aaa gta gta gtt aaa     1056Ala Ser Thr Pro Phe Glu Asn Asn Thr Glu Ty #r Lys Val Val Val Lys            310       #           315       #           320ggt att aaa gac aaa aat ggc aaa gaa ttt aa#a gaa gat gca ttc act     1104Gly Ile Lys Asp Lys Asn Gly Lys Glu Phe Ly #s Glu Asp Ala Phe Thr        325           #       330           #       335ttc aag ctt cga aat gat gct gta gtt act ca#a gtg ttt gga act aat     1152Phe Lys Leu Arg Asn Asp Ala Val Val Thr Gl #n Val Phe Gly Thr Asn    340               #   345               #   350gta aca aac aac act tct gta aac tta gca gc#a ggt act ttc gac act     1200Val Thr Asn Asn Thr Ser Val Asn Leu Ala Al #a Gly Thr Phe Asp Thr355                 3 #60                 3 #65                 3 #70gac gat act tta aca gta gta ttt gat aag tt#g tta gca cct gaa act     1248Asp Asp Thr Leu Thr Val Val Phe Asp Lys Le #u Leu Ala Pro Glu Thr                375   #               380   #               385gta aac agc tcg aac gtt act att aca gat gt#t gaa act gga aaa cgc     1296Val Asn Ser Ser Asn Val Thr Ile Thr Asp Va #l Glu Thr Gly Lys Arg            390       #           395       #           400att cca gta att gca tct act tct ggt tct ac#a att act att acg tta     1344Ile Pro Val Ile Ala Ser Thr Ser Gly Ser Th #r Ile Thr Ile Thr Leu        405           #       410           #       415aaa gaa gcg tta gta act ggt aaa caa tat aa#a ctt gct atc aat aat     1392Lys Glu Ala Leu Val Thr Gly Lys Gln Tyr Ly #s Leu Ala Ile Asn Asn    420               #   425               #   430gtt aaa aca tta act ggt tac aat gca gaa gc#t tac gag tta gtg ttc     1440Val Lys Thr Leu Thr Gly Tyr Asn Ala Glu Al #a Tyr Glu Leu Val Phe435                 4 #40                 4 #45                 4 #50act gca aac gca tca gca cca act gtt gct ac#c gct cct act act tta     1488Thr Ala Asn Ala Ser Ala Pro Thr Val Ala Th #r Ala Pro Thr Thr Leu                455   #               460   #               465ggt ggt aca act tta tct act ggt tct ctt ac#a aca aat gtt tgg ggt     1536Gly Gly Thr Thr Leu Ser Thr Gly Ser Leu Th #r Thr Asn Val Trp Gly            470       #           475       #           480aaa ttg gct ggt ggt gtg aat gaa gct gga ac#t tat tat cct ggt ctt     1584Lys Leu Ala Gly Gly Val Asn Glu Ala Gly Th #r Tyr Tyr Pro Gly Leu        485           #       490           #       495caa ttc aca aca acg ttt gct act aag tta ga#c gaa tct act tta gct     1632Gln Phe Thr Thr Thr Phe Ala Thr Lys Leu As #p Glu Ser Thr Leu Ala    500               #   505               #   510gat aac ttt gta tta gtt gaa aaa gaa tct gg#t aca gtt gtt gct tct     1680Asp Asn Phe Val Leu Val Glu Lys Glu Ser Gl #y Thr Val Val Ala Ser515                 5 #20                 5 #25                 5 #30gaa cta aaa tat aat gca gac gct aaa atg gt#a act tta gtg cca aaa     1728Glu Leu Lys Tyr Asn Ala Asp Ala Lys Met Va #l Thr Leu Val Pro Lys                535   #               540   #               545gcg gac ctt aaa gaa aat aca atc tat caa at#c aaa att aaa aaa ggc     1776Ala Asp Leu Lys Glu Asn Thr Ile Tyr Gln Il #e Lys Ile Lys Lys Gly            550       #           555       #           560ttg aag tcc gat aaa ggt att gaa tta ggc ac#t gtt aac gag aaa aca     1824Leu Lys Ser Asp Lys Gly Ile Glu Leu Gly Th #r Val Asn Glu Lys Thr        565           #       570           #       575tat gag ttc aaa act caa gac tta act gct cc#t aca gtt att agc gta     1872Tyr Glu Phe Lys Thr Gln Asp Leu Thr Ala Pr #o Thr Val Ile Ser Val    580               #   585               #   590acg tct aaa aat ggc gac gct gga tta aaa gt#a act gaa gct caa gaa     1920Thr Ser Lys Asn Gly Asp Ala Gly Leu Lys Va #l Thr Glu Ala Gln Glu595                 6 #00                 6 #05                 6 #10ttt act gtg aag ttc tca gag aat tta aat ac#a ttt aat gct aca acc     1968Phe Thr Val Lys Phe Ser Glu Asn Leu Asn Th #r Phe Asn Ala Thr Thr                615   #               620   #               625gtt tcg ggt agc aca atc aca tac ggt caa gt#t gct gta gta aaa gcg     2016Val Ser Gly Ser Thr Ile Thr Tyr Gly Gln Va #l Ala Val Val Lys Ala            630       #           635       #           640ggt gca aac tta tct gct ctt aca gca agt ga#c atc att cca gct agt     2064Gly Ala Asn Leu Ser Ala Leu Thr Ala Ser As #p Ile Ile Pro Ala Ser        645           #       650           #       655gtt gaa gcg gtt act ggt caa gat gga aca ta#c aaa gtg aaa gtt gct     2112Val Glu Ala Val Thr Gly Gln Asp Gly Thr Ty #r Lys Val Lys Val Ala    660               #   665               #   670gct aac caa tta gaa cgt aac caa ggg tac aa#a tta gta gtg ttc ggt     2160Ala Asn Gln Leu Glu Arg Asn Gln Gly Tyr Ly #s Leu Val Val Phe Gly675                 6 #80                 6 #85                 6 #90aaa ggt gca aca gct cct gtt aaa gat gct gc#a aat gca aat act tta     2208Lys Gly Ala Thr Ala Pro Val Lys Asp Ala Al #a Asn Ala Asn Thr Leu                695   #               700   #               705gca act aac tat atc tat aca ttt aca act ga#a ggt caa gac gta aca     2256Ala Thr Asn Tyr Ile Tyr Thr Phe Thr Thr Gl #u Gly Gln Asp Val Thr            710       #           715       #           720gca cca acg gtt aca aaa gta ttc aaa ggt ga#t tct tta aaa gac gct     2304Ala Pro Thr Val Thr Lys Val Phe Lys Gly As #p Ser Leu Lys Asp Ala        725           #       730           #       735gat gca gtt act aca ctt acg aac gtt gat gc#a ggt caa aaa ttc act     2352Asp Ala Val Thr Thr Leu Thr Asn Val Asp Al #a Gly Gln Lys Phe Thr    740               #   745               #   750atc caa ttt agc gaa gaa tta aaa act tct ag#t ggt tct tta gtg ggt     2400Ile Gln Phe Ser Glu Glu Leu Lys Thr Ser Se #r Gly Ser Leu Val Gly755                 7 #60                 7 #65                 7 #70ggc aaa gta act gtc gag aaa tta aca aac aa#c gga tgg gta gat gct     2448Gly Lys Val Thr Val Glu Lys Leu Thr Asn As #n Gly Trp Val Asp Ala                775   #               780   #               785ggt act gga aca act gta tca gtt gct cct aa#g aca gat gca aat ggt     2496Gly Thr Gly Thr Thr Val Ser Val Ala Pro Ly #s Thr Asp Ala Asn Gly            790       #           795       #           800aaa gta aca gct gct gtg gtt aca tta act gg#t ctt gac aat aac gac     2544Lys Val Thr Ala Ala Val Val Thr Leu Thr Gl #y Leu Asp Asn Asn Asp        805           #       810           #       815aaa gat gcg aaa ttg cgt ctg gta gta gat aa#g tct tct act gat gga     2592Lys Asp Ala Lys Leu Arg Leu Val Val Asp Ly #s Ser Ser Thr Asp Gly    820               #   825               #   830att gct gat gta gct ggt aat gta att aag ga#a aaa gat att tta att     2640Ile Ala Asp Val Ala Gly Asn Val Ile Lys Gl #u Lys Asp Ile Leu Ile835                 8 #40                 8 #45                 8 #50cgt tac aac agc tgg aga cac act gta gct tc#t gtg aaa gct gct gct     2688Arg Tyr Asn Ser Trp Arg His Thr Val Ala Se #r Val Lys Ala Ala Ala                855   #               860   #               865gac aaa gat ggt caa aac gct tct gct gca tt#c cca aca agc act gca     2736Asp Lys Asp Gly Gln Asn Ala Ser Ala Ala Ph #e Pro Thr Ser Thr Ala            870       #           875       #           880att gat aca act aag agc tta tta gtt gaa tt#c aat gaa act gat tta     2784Ile Asp Thr Thr Lys Ser Leu Leu Val Glu Ph #e Asn Glu Thr Asp Leu        885           #       890           #       895gcg gaa gtt aaa cct gag aac atc gtt gtt aa#a gat gca gca ggt aat     2832Ala Glu Val Lys Pro Glu Asn Ile Val Val Ly #s Asp Ala Ala Gly Asn    900               #   905               #   910gcg gta gct ggt act gta aca gca tta gac gg#t tct aca aat aaa ttt     2880Ala Val Ala Gly Thr Val Thr Ala Leu Asp Gl #y Ser Thr Asn Lys Phe915                 9 #20                 9 #25                 9 #30gta ttc act cca tct caa gaa tta aaa gct gg#t aca gtt tac tct gta     2928Val Phe Thr Pro Ser Gln Glu Leu Lys Ala Gl #y Thr Val Tyr Ser Val                935   #               940   #               945aca att gac ggt gtg aga gat aaa gta ggt aa#c aca atc tct aaa tac     2976Thr Ile Asp Gly Val Arg Asp Lys Val Gly As #n Thr Ile Ser Lys Tyr            950       #           955       #           960att act tcg ttc aag act gta tct gcg aat cc#a acg tta tct tca atc     3024Ile Thr Ser Phe Lys Thr Val Ser Ala Asn Pr #o Thr Leu Ser Ser Ile        965           #       970           #       975agc att gct gac ggt gca gtt aac gtt gac cg#t tct aaa aca att aca     3072Ser Ile Ala Asp Gly Ala Val Asn Val Asp Ar #g Ser Lys Thr Ile Thr    980               #   985               #   990att gaa ttc agc gat tca gtt cca aac cca ac#a atc act ctt aag aag     3120Ile Glu Phe Ser Asp Ser Val Pro Asn Pro Th #r Ile Thr Leu Lys Lys995                1000  #               1005   #              1010gct gac gga act tca ttt act aat tac act tt#a gta aat gta aat aat     3168Ala Asp Gly Thr Ser Phe Thr Asn Tyr Thr Le #u Val Asn Val Asn Asn               1015   #              1020    #             1025gaa aat aaa aca tac aaa att gta ttc cac aa#a ggt gta aca ctt gac     3216Glu Asn Lys Thr Tyr Lys Ile Val Phe His Ly #s Gly Val Thr Leu Asp           1030       #          1035        #         1040gag ttt act caa tat gag tta gca gtt tca aa#a gat ttt caa act ggt     3264Glu Phe Thr Gln Tyr Glu Leu Ala Val Ser Ly #s Asp Phe Gln Thr Gly       1045           #      1050            #     1055act gat att gat agc aaa gtt aca ttc atc ac#a ggt tct gtt gct act     3312Thr Asp Ile Asp Ser Lys Val Thr Phe Ile Th #r Gly Ser Val Ala Thr   1060               #  1065                # 1070gac gaa gta aaa cct gct cta gta ggc gtt gg#t tca tgg aat gga aca     3360Asp Glu Val Lys Pro Ala Leu Val Gly Val Gl #y Ser Trp Asn Gly Thr1075               1080  #               1085   #              1090agc tat act cag gat gct gca gca aca cga ct#t cgg tct gta gct gac     3408Ser Tyr Thr Gln Asp Ala Ala Ala Thr Arg Le #u Arg Ser Val Ala Asp               1095   #              1100    #             1105ttc gtt gcg gag cca gtt gcc ctt caa ttc tc#a gaa ggt atc gat tta     3456Phe Val Ala Glu Pro Val Ala Leu Gln Phe Se #r Glu Gly Ile Asp Leu           1110       #          1115        #         1120acg aat gca act gtg aca gta aca aat att ac#t gat gat aaa act gtt     3504Thr Asn Ala Thr Val Thr Val Thr Asn Ile Th #r Asp Asp Lys Thr Val       1125           #      1130            #     1135gaa gtt att tca aaa gag agt gta gac gca ga#c cat gat gca ggt gct     3552Glu Val Ile Ser Lys Glu Ser Val Asp Ala As #p His Asp Ala Gly Ala   1140               #  1145                # 1150act aag gag aca tta gta att aac aca gtt ac#t cct tta gta ctt gat     3600Thr Lys Glu Thr Leu Val Ile Asn Thr Val Th #r Pro Leu Val Leu Asp1155               1160  #               1165   #              1170aac agc aag act tat aag att gtt gta agt gg#a gtt aaa gat gca gca     3648Asn Ser Lys Thr Tyr Lys Ile Val Val Ser Gl #y Val Lys Asp Ala Ala               1175   #              1180    #             1185ggt aat gtt gca gat act att aca ttc tat at #t aag taa              #   3687 Gly Asn Val Ala Asp Thr Ile Thr Phe Tyr Il #e Lys           1190       #          1195 <210> SEQ ID NO 2<211> LENGTH: 1228 <212> TYPE: PRT<213> ORGANISM: Bacillus stearothermophilus <400> SEQUENCE: 2Met Asp Arg Lys Lys Ala Val Lys Leu Ala Th #r Ala Ser Ala Ile Ala-30                 - #25                 - #20                 - #15Ala Ser Ala Phe Val Ala Ala Asn Pro Asn Al #a Ser Glu Ala Ala Thr                -10   #                -5   #            -1   1Asp Val Ala Thr Val Val Ser Gln Ala Lys Al #a Gln Phe Lys Lys Ala          5         #          10         #          15Tyr Tyr Thr Tyr Ser His Thr Val Thr Glu Th #r Gly Glu Phe Pro Asn     20              #     25              #     30Ile Asn Asp Val Tyr Ala Glu Tyr Asn Lys Al #a Lys Lys Arg Tyr Arg 35                  # 40                  # 45                  # 50Asp Ala Val Ala Leu Val Asn Lys Ala Gly Gl #y Ala Lys Lys Asp Ala                 55  #                 60  #                 65Tyr Leu Ala Asp Leu Gln Lys Glu Tyr Glu Th #r Tyr Val Phe Lys Ala             70      #             75      #             80Asn Pro Lys Ser Gly Glu Ala Arg Val Ala Th #r Tyr Ile Asp Ala Tyr         85          #         90          #         95Asn Tyr Ala Thr Lys Leu Asp Glu Met Arg Gl #n Glu Leu Glu Ala Ala    100               #   105               #   110Val Gln Ala Lys Asp Leu Glu Lys Ala Glu Gl #n Tyr Tyr His Lys Ile115                 1 #20                 1 #25                 1 #30Pro Tyr Glu Ile Lys Thr Arg Thr Val Ile Le #u Asp Arg Val Tyr Gly                135   #               140   #               145Lys Thr Thr Arg Asp Leu Leu Arg Ser Thr Ph #e Lys Ala Lys Ala Gln            150       #           155       #           160Glu Leu Arg Asp Ser Leu Ile Tyr Asp Ile Th #r Val Ala Met Lys Ala        165           #       170           #       175Arg Glu Val Gln Asp Ala Val Lys Ala Gly As #n Leu Asp Lys Ala Lys    180               #   185               #   190Ala Ala Val Asp Gln Ile Asn Gln Tyr Leu Pr #o Lys Val Thr Asp Ala195                 2 #00                 2 #05                 2 #10Phe Lys Thr Glu Leu Thr Glu Val Ala Lys Ly #s Ala Leu Asp Ala Asp                215   #               220   #               225Glu Ala Ala Leu Thr Pro Lys Val Glu Ser Va #l Ser Ala Ile Asn Thr            230       #           235       #           240Gln Asn Lys Ala Val Glu Leu Thr Ala Val Pr #o Val Asn Gly Thr Leu        245           #       250           #       255Lys Leu Gln Leu Ser Ala Ala Ala Asn Glu As #p Thr Val Asn Val Asn    260               #   265               #   270Thr Val Arg Ile Tyr Lys Val Asp Gly Asn Il #e Pro Phe Ala Leu Asn275                 2 #80                 2 #85                 2 #90Thr Ala Asp Val Ser Leu Ser Thr Asp Gly Ly #s Thr Ile Thr Val Asp                295   #               300   #               305Ala Ser Thr Pro Phe Glu Asn Asn Thr Glu Ty #r Lys Val Val Val Lys            310       #           315       #           320Gly Ile Lys Asp Lys Asn Gly Lys Glu Phe Ly #s Glu Asp Ala Phe Thr        325           #       330           #       335Phe Lys Leu Arg Asn Asp Ala Val Val Thr Gl #n Val Phe Gly Thr Asn    340               #   345               #   350Val Thr Asn Asn Thr Ser Val Asn Leu Ala Al #a Gly Thr Phe Asp Thr355                 3 #60                 3 #65                 3 #70Asp Asp Thr Leu Thr Val Val Phe Asp Lys Le #u Leu Ala Pro Glu Thr                375   #               380   #               385Val Asn Ser Ser Asn Val Thr Ile Thr Asp Va #l Glu Thr Gly Lys Arg            390       #           395       #           400Ile Pro Val Ile Ala Ser Thr Ser Gly Ser Th #r Ile Thr Ile Thr Leu        405           #       410           #       415Lys Glu Ala Leu Val Thr Gly Lys Gln Tyr Ly #s Leu Ala Ile Asn Asn    420               #   425               #   430Val Lys Thr Leu Thr Gly Tyr Asn Ala Glu Al #a Tyr Glu Leu Val Phe435                 4 #40                 4 #45                 4 #50Thr Ala Asn Ala Ser Ala Pro Thr Val Ala Th #r Ala Pro Thr Thr Leu                455   #               460   #               465Gly Gly Thr Thr Leu Ser Thr Gly Ser Leu Th #r Thr Asn Val Trp Gly            470       #           475       #           480Lys Leu Ala Gly Gly Val Asn Glu Ala Gly Th #r Tyr Tyr Pro Gly Leu        485           #       490           #       495Gln Phe Thr Thr Thr Phe Ala Thr Lys Leu As #p Glu Ser Thr Leu Ala    500               #   505               #   510Asp Asn Phe Val Leu Val Glu Lys Glu Ser Gl #y Thr Val Val Ala Ser515                 5 #20                 5 #25                 5 #30Glu Leu Lys Tyr Asn Ala Asp Ala Lys Met Va #l Thr Leu Val Pro Lys                535   #               540   #               545Ala Asp Leu Lys Glu Asn Thr Ile Tyr Gln Il #e Lys Ile Lys Lys Gly            550       #           555       #           560Leu Lys Ser Asp Lys Gly Ile Glu Leu Gly Th #r Val Asn Glu Lys Thr        565           #       570           #       575Tyr Glu Phe Lys Thr Gln Asp Leu Thr Ala Pr #o Thr Val Ile Ser Val    580               #   585               #   590Thr Ser Lys Asn Gly Asp Ala Gly Leu Lys Va #l Thr Glu Ala Gln Glu595                 6 #00                 6 #05                 6 #10Phe Thr Val Lys Phe Ser Glu Asn Leu Asn Th #r Phe Asn Ala Thr Thr                615   #               620   #               625Val Ser Gly Ser Thr Ile Thr Tyr Gly Gln Va #l Ala Val Val Lys Ala            630       #           635       #           640Gly Ala Asn Leu Ser Ala Leu Thr Ala Ser As #p Ile Ile Pro Ala Ser        645           #       650           #       655Val Glu Ala Val Thr Gly Gln Asp Gly Thr Ty #r Lys Val Lys Val Ala    660               #   665               #   670Ala Asn Gln Leu Glu Arg Asn Gln Gly Tyr Ly #s Leu Val Val Phe Gly675                 6 #80                 6 #85                 6 #90Lys Gly Ala Thr Ala Pro Val Lys Asp Ala Al #a Asn Ala Asn Thr Leu                695   #               700   #               705Ala Thr Asn Tyr Ile Tyr Thr Phe Thr Thr Gl #u Gly Gln Asp Val Thr            710       #           715       #           720Ala Pro Thr Val Thr Lys Val Phe Lys Gly As #p Ser Leu Lys Asp Ala        725           #       730           #       735Asp Ala Val Thr Thr Leu Thr Asn Val Asp Al #a Gly Gln Lys Phe Thr    740               #   745               #   750Ile Gln Phe Ser Glu Glu Leu Lys Thr Ser Se #r Gly Ser Leu Val Gly755                 7 #60                 7 #65                 7 #70Gly Lys Val Thr Val Glu Lys Leu Thr Asn As #n Gly Trp Val Asp Ala                775   #               780   #               785Gly Thr Gly Thr Thr Val Ser Val Ala Pro Ly #s Thr Asp Ala Asn Gly            790       #           795       #           800Lys Val Thr Ala Ala Val Val Thr Leu Thr Gl #y Leu Asp Asn Asn Asp        805           #       810           #       815Lys Asp Ala Lys Leu Arg Leu Val Val Asp Ly #s Ser Ser Thr Asp Gly    820               #   825               #   830Ile Ala Asp Val Ala Gly Asn Val Ile Lys Gl #u Lys Asp Ile Leu Ile835                 8 #40                 8 #45                 8 #50Arg Tyr Asn Ser Trp Arg His Thr Val Ala Se #r Val Lys Ala Ala Ala                855   #               860   #               865Asp Lys Asp Gly Gln Asn Ala Ser Ala Ala Ph #e Pro Thr Ser Thr Ala            870       #           875       #           880Ile Asp Thr Thr Lys Ser Leu Leu Val Glu Ph #e Asn Glu Thr Asp Leu        885           #       890           #       895Ala Glu Val Lys Pro Glu Asn Ile Val Val Ly #s Asp Ala Ala Gly Asn    900               #   905               #   910Ala Val Ala Gly Thr Val Thr Ala Leu Asp Gl #y Ser Thr Asn Lys Phe915                 9 #20                 9 #25                 9 #30Val Phe Thr Pro Ser Gln Glu Leu Lys Ala Gl #y Thr Val Tyr Ser Val                935   #               940   #               945Thr Ile Asp Gly Val Arg Asp Lys Val Gly As #n Thr Ile Ser Lys Tyr            950       #           955       #           960Ile Thr Ser Phe Lys Thr Val Ser Ala Asn Pr #o Thr Leu Ser Ser Ile        965           #       970           #       975Ser Ile Ala Asp Gly Ala Val Asn Val Asp Ar #g Ser Lys Thr Ile Thr    980               #   985               #   990Ile Glu Phe Ser Asp Ser Val Pro Asn Pro Th #r Ile Thr Leu Lys Lys995                1000  #               1005   #              1010Ala Asp Gly Thr Ser Phe Thr Asn Tyr Thr Le #u Val Asn Val Asn Asn               1015   #              1020    #             1025Glu Asn Lys Thr Tyr Lys Ile Val Phe His Ly #s Gly Val Thr Leu Asp           1030       #          1035        #         1040Glu Phe Thr Gln Tyr Glu Leu Ala Val Ser Ly #s Asp Phe Gln Thr Gly       1045           #      1050            #     1055Thr Asp Ile Asp Ser Lys Val Thr Phe Ile Th #r Gly Ser Val Ala Thr   1060               #  1065                # 1070Asp Glu Val Lys Pro Ala Leu Val Gly Val Gl #y Ser Trp Asn Gly Thr1075               1080  #               1085   #              1090Ser Tyr Thr Gln Asp Ala Ala Ala Thr Arg Le #u Arg Ser Val Ala Asp               1095   #              1100    #             1105Phe Val Ala Glu Pro Val Ala Leu Gln Phe Se #r Glu Gly Ile Asp Leu           1110       #          1115        #         1120Thr Asn Ala Thr Val Thr Val Thr Asn Ile Th #r Asp Asp Lys Thr Val       1125           #      1130            #     1135Glu Val Ile Ser Lys Glu Ser Val Asp Ala As #p His Asp Ala Gly Ala   1140               #  1145                # 1150Thr Lys Glu Thr Leu Val Ile Asn Thr Val Th #r Pro Leu Val Leu Asp1155               1160  #               1165   #              1170Asn Ser Lys Thr Tyr Lys Ile Val Val Ser Gl #y Val Lys Asp Ala Ala               1175   #              1180    #             1185Gly Asn Val Ala Asp Thr Ile Thr Phe Tyr Il #e Lys            1190      #          1195 <210> SEQ ID NO 3 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      primer <400> SEQUENCE: 3ttaatcgatt ctagatggat aggaaaaaag ctg        #                  #         33 <210> SEQ ID NO 4 <211> LENGTH: 37 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      primer <400> SEQUENCE: 4atacccgggg gtacggatcc gatacagatt tgagcaa       #                  #      37 <210> SEQ ID NO 5 <211> LENGTH: 2766 <212> TYPE: DNA<213> ORGANISM: Bacillus stearothermophilus <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(2763)<221> NAME/KEY: sig_peptide <222> LOCATION: (1)..(93)<221> NAME/KEY: mat_peptide <222> LOCATION: (94)..(2763)<400> SEQUENCE: 5 atg gct tat caa cct aag tct ttt cgc aag tt#t gtt gcg aca act gca       48Met Ala Tyr Gln Pro Lys Ser Phe Arg Lys Ph #e Val Ala Thr Thr Ala    -30               #   -25               #   -20aca gct gcc att gta gca tct gcg gta gct cc#t gta gta tct gca gca       96Thr Ala Ala Ile Val Ala Ser Ala Val Ala Pr #o Val Val Ser Ala Ala-15                 - #10                   #-5              -1    #1agc ttc aca gat gtt gcg ccg caa tat aaa ga#t gcg atc gat ttc tta      144Ser Phe Thr Asp Val Ala Pro Gln Tyr Lys As #p Ala Ile Asp Phe Leu              5     #              10     #              15gta tca act ggt gca aca aaa ggt aaa aca ga#a aca aaa ttc ggc gtt      192Val Ser Thr Gly Ala Thr Lys Gly Lys Thr Gl #u Thr Lys Phe Gly Val         20          #         25          #         30tac gat gaa atc act cgt cta gat gcg gca gt#t att ctt gca aga gta      240Tyr Asp Glu Ile Thr Arg Leu Asp Ala Ala Va #l Ile Leu Ala Arg Val     35              #     40              #     45tta aaa cta gac gtt gac aac gca aaa gac gc#a ggc ttc aca gat gtg      288Leu Lys Leu Asp Val Asp Asn Ala Lys Asp Al #a Gly Phe Thr Asp Val 50                  # 55                  # 60                  # 65cca aaa gac cgt gca aaa tac gtc aac gcg ct#t gta gaa gct ggc gta      336Pro Lys Asp Arg Ala Lys Tyr Val Asn Ala Le #u Val Glu Ala Gly Val                 70  #                 75  #                 80tta aac ggt aaa gca cct ggc aaa ttt ggt gc#a tac gac cca tta act      384Leu Asn Gly Lys Ala Pro Gly Lys Phe Gly Al #a Tyr Asp Pro Leu Thr             85      #             90      #             95cgc gtt gaa atg gca aaa atc atc gcg aac cg#t tac aaa tta aaa gct      432Arg Val Glu Met Ala Lys Ile Ile Ala Asn Ar #g Tyr Lys Leu Lys Ala        100           #       105           #       110gac gat gta aaa ctt cca ttc act gat gta aa#c gat aca tgg gca cca      480Asp Asp Val Lys Leu Pro Phe Thr Asp Val As #n Asp Thr Trp Ala Pro    115               #   120               #   125tac gta aaa gcg ctt tat aaa tac gaa gta ac#c aaa agg tta aaa cac      528Tyr Val Lys Ala Leu Tyr Lys Tyr Glu Val Th #r Lys Arg Leu Lys His130                 1 #35                 1 #40                 1 #45caa caa gct tcg gtg cat acc aaa aac atc ac#t ctg cgt gac ttt gcg      576Gln Gln Ala Ser Val His Thr Lys Asn Ile Th #r Leu Arg Asp Phe Ala                150   #               155   #               160caa ttt gta tat aga gcg gtg aat att aat gc#a gtg cca gaa ata gtt      624Gln Phe Val Tyr Arg Ala Val Asn Ile Asn Al #a Val Pro Glu Ile Val            165       #           170       #           175gaa gta act gcg gtt aat tcg act aca gtg aa#a gta aca ttc aat acg      672Glu Val Thr Ala Val Asn Ser Thr Thr Val Ly #s Val Thr Phe Asn Thr        180           #       185           #       190caa att gct gat gtt gat ttc aca aat ttt gc#t atc gat aac ggt tta      720Gln Ile Ala Asp Val Asp Phe Thr Asn Phe Al #a Ile Asp Asn Gly Leu    195               #   200               #   205act gtt act aaa gca act ctt tct cgt gat aa#a aaa tcc gta gag gtt      768Thr Val Thr Lys Ala Thr Leu Ser Arg Asp Ly #s Lys Ser Val Glu Val210                 2 #15                 2 #20                 2 #25gtg gta aat aaa ccg ttt act cgt aat cag ga#a tat aca att aca gcg      816Val Val Asn Lys Pro Phe Thr Arg Asn Gln Gl #u Tyr Thr Ile Thr Ala                230   #               235   #               240aca ggc att aaa aat tta aaa ggc gag acc gc#t aag gaa tta act ggt      864Thr Gly Ile Lys Asn Leu Lys Gly Glu Thr Al #a Lys Glu Leu Thr Gly            245       #           250       #           255aag ttt gtt tgg tct gtt caa gat gcg gta ac#t gtt gca cta aat aat      912Lys Phe Val Trp Ser Val Gln Asp Ala Val Th #r Val Ala Leu Asn Asn        260           #       265           #       270agt tcg ctt aaa gtt gga gag gaa tct ggt tt#a act gta aaa gat cag      960Ser Ser Leu Lys Val Gly Glu Glu Ser Gly Le #u Thr Val Lys Asp Gln    275               #   280               #   285gat ggc aaa gat gtt gta ggt gct aaa gta ga#a ctt act tct tct aat     1008Asp Gly Lys Asp Val Val Gly Ala Lys Val Gl #u Leu Thr Ser Ser Asn290                 2 #95                 3 #00                 3 #05act aat att gtt gta gtt tca agt ggc gaa gt#a tca gta tct gct gct     1056Thr Asn Ile Val Val Val Ser Ser Gly Glu Va #l Ser Val Ser Ala Ala                310   #               315   #               320aaa gtt aca gct gta aaa ccg gga aca gct ga#t gtt act gca aaa gtt     1104Lys Val Thr Ala Val Lys Pro Gly Thr Ala As #p Val Thr Ala Lys Val            325       #           330       #           335aca tta cca gat ggt gtt gta cta aca aat ac#a ttt aaa gtg aca gtt     1152Thr Leu Pro Asp Gly Val Val Leu Thr Asn Th #r Phe Lys Val Thr Val        340           #       345           #       350aca gaa gtg cct gtt caa gtc caa aat caa gg#a ttt act tta gtt gat     1200Thr Glu Val Pro Val Gln Val Gln Asn Gln Gl #y Phe Thr Leu Val Asp    355               #   360               #   365aat ctt tct aat gct cca cag aat aca gtt gc#a ttt aac aaa gct gag     1248Asn Leu Ser Asn Ala Pro Gln Asn Thr Val Al #a Phe Asn Lys Ala Glu370                 3 #75                 3 #80                 3 #85aaa gta act tca atg ttt gct gga gaa act aa#a aca gtt gca atg tat     1296Lys Val Thr Ser Met Phe Ala Gly Glu Thr Ly #s Thr Val Ala Met Tyr                390   #               395   #               400gat act aaa aac ggt gat cct gaa act aaa cc#t gtt gat ttc aaa gat     1344Asp Thr Lys Asn Gly Asp Pro Glu Thr Lys Pr #o Val Asp Phe Lys Asp            405       #           410       #           415gca act gta cgt tca tta aat cca att att gc#a aca gct gct att aat     1392Ala Thr Val Arg Ser Leu Asn Pro Ile Ile Al #a Thr Ala Ala Ile Asn        420           #       425           #       430ggt agt gag ctc ctt gtc aca gct aat gct gg#c caa tct gga aaa gct     1440Gly Ser Glu Leu Leu Val Thr Ala Asn Ala Gl #y Gln Ser Gly Lys Ala    435               #   440               #   445tca ttt gaa gta aca tta aaa gat aat aca aa#a aga aca ttt aca gtt     1488Ser Phe Glu Val Thr Leu Lys Asp Asn Thr Ly #s Arg Thr Phe Thr Val450                 4 #55                 4 #60                 4 #65gat gta aaa aaa gac cct gta tta caa gat at#a aaa gta gat gca act     1536Asp Val Lys Lys Asp Pro Val Leu Gln Asp Il #e Lys Val Asp Ala Thr                470   #               475   #               480tct gtt aaa ctt tcc gat gaa gct gtt ggc gg#c ggg gaa gtt gaa gga     1584Ser Val Lys Leu Ser Asp Glu Ala Val Gly Gl #y Gly Glu Val Glu Gly            485       #           490       #           495gtt aac caa aaa acg att aaa gta agt gca gt#t gac caa tac ggt aaa     1632Val Asn Gln Lys Thr Ile Lys Val Ser Ala Va #l Asp Gln Tyr Gly Lys        500           #       505           #       510gaa att aaa ttt ggt aca aaa ggt aaa gtt ac#t gtt aca act aat aca     1680Glu Ile Lys Phe Gly Thr Lys Gly Lys Val Th #r Val Thr Thr Asn Thr    515               #   520               #   525gaa gga cta gtt att aaa aat gta aat agc ga#t aat aca att gac ttt     1728Glu Gly Leu Val Ile Lys Asn Val Asn Ser As #p Asn Thr Ile Asp Phe530                 5 #35                 5 #40                 5 #45gat agc ggc aat agt gca act gac caa ttt gt#t gtc gtt gca aca aaa     1776Asp Ser Gly Asn Ser Ala Thr Asp Gln Phe Va #l Val Val Ala Thr Lys                550   #               555   #               560gac aaa att gtc aat ggt aaa gta gaa gtt aa#a tat ttc aaa aat gct     1824Asp Lys Ile Val Asn Gly Lys Val Glu Val Ly #s Tyr Phe Lys Asn Ala            565       #           570       #           575agt gac aca aca cca act tca act aaa aca at#t act gtt aat gta gta     1872Ser Asp Thr Thr Pro Thr Ser Thr Lys Thr Il #e Thr Val Asn Val Val        580           #       585           #       590aat gta aaa gct gac gct aca cca gta gga tt#a gat att gta gca cct     1920Asn Val Lys Ala Asp Ala Thr Pro Val Gly Le #u Asp Ile Val Ala Pro    595               #   600               #   605tct aaa att gat gta aat gct cca aac act gc#t tct act gca gat gtt     1968Ser Lys Ile Asp Val Asn Ala Pro Asn Thr Al #a Ser Thr Ala Asp Val610                 6 #15                 6 #20                 6 #25gat ttt ata aat ttc gaa agt gtt gag att ta#c aca ctc gat tca aat     2016Asp Phe Ile Asn Phe Glu Ser Val Glu Ile Ty #r Thr Leu Asp Ser Asn                630   #               635   #               640ggt aga cgt caa aaa aaa gtt act cca act gc#a act aca ctt gta ggt     2064Gly Arg Arg Gln Lys Lys Val Thr Pro Thr Al #a Thr Thr Leu Val Gly            645       #           650       #           655aca aaa aaa aaa aaa aaa gtt aat ggg aat gt#a tta caa ttc aag ggg     2112Thr Lys Lys Lys Lys Lys Val Asn Gly Asn Va #l Leu Gln Phe Lys Gly        660           #       665           #       670aac gaa gaa tta acg cta tca act tct tct ag#t aca gga aac gta gat     2160Asn Glu Glu Leu Thr Leu Ser Thr Ser Ser Se #r Thr Gly Asn Val Asp    675               #   680               #   685gga aca gca gaa gga atg aca aaa cgt att cc#a ggg aaa tat atc aac     2208Gly Thr Ala Glu Gly Met Thr Lys Arg Ile Pr #o Gly Lys Tyr Ile Asn690                 6 #95                 7 #00                 7 #05tct gca agt gta cct gcc agt gca aca gta gc#a aca agt cct gtt act     2256Ser Ala Ser Val Pro Ala Ser Ala Thr Val Al #a Thr Ser Pro Val Thr                710   #               715   #               720gta aag ctt aat tca agt gat aat gat tta ac#a ttt gaa gaa tta ata     2304Val Lys Leu Asn Ser Ser Asp Asn Asp Leu Th #r Phe Glu Glu Leu Ile            725       #           730       #           735ttc ggt gta att gac cct aca caa tta gtc aa#a gat gaa gac atc aac     2352Phe Gly Val Ile Asp Pro Thr Gln Leu Val Ly #s Asp Glu Asp Ile Asn        740           #       745           #       750gaa ttt att gca gtt tca aaa gcg gct aaa aa#t gat gga tat ttg tat     2400Glu Phe Ile Ala Val Ser Lys Ala Ala Lys As #n Asp Gly Tyr Leu Tyr    755               #   760               #   765aat aaa ccg ctt gta acg gtt aaa gat gca tc#a gga aaa gtt att cca     2448Asn Lys Pro Leu Val Thr Val Lys Asp Ala Se #r Gly Lys Val Ile Pro770                 7 #75                 7 #80                 7 #85aca ggt gca aat gtt tac ggt cta aat cat ga#t gca act aac gga aac     2496Thr Gly Ala Asn Val Tyr Gly Leu Asn His As #p Ala Thr Asn Gly Asn                790   #               795   #               800att tgg ttt gat gag gaa caa gct ggc tta gc#t aaa aaa ttt agt gat     2544Ile Trp Phe Asp Glu Glu Gln Ala Gly Leu Al #a Lys Lys Phe Ser Asp            805       #           810       #           815gta cat ttt gat gtt gat ttt tca tta act aa#c gtt gta aaa act ggt     2592Val His Phe Asp Val Asp Phe Ser Leu Thr As #n Val Val Lys Thr Gly        820           #       825           #       830agc ggt aca gtt tct tca tcg cca tca tta tc#t gac gca att caa ctt     2640Ser Gly Thr Val Ser Ser Ser Pro Ser Leu Se #r Asp Ala Ile Gln Leu    835               #   840               #   845act aat tca ggc gat gca gta tcg ttt aca tt#a gtt atc aaa tca att     2688Thr Asn Ser Gly Asp Ala Val Ser Phe Thr Le #u Val Ile Lys Ser Ile850                 8 #55                 8 #60                 8 #65tat gtt aaa ggc gca gat aaa gat gat aat aa#c tta ctt gca gcc cct     2736Tyr Val Lys Gly Ala Asp Lys Asp Asp Asn As #n Leu Leu Ala Ala Pro                870   #               875   #               880gtt tct gtc aat gtg act gtg aca aaa taa   #                  #         2766 Val Ser Val Asn Val Thr Val Thr Lys             885      #           890 <210> SEQ ID NO 6 <211> LENGTH: 921 <212> TYPE: PRT<213> ORGANISM: Bacillus stearothermophilus <400> SEQUENCE: 6Met Ala Tyr Gln Pro Lys Ser Phe Arg Lys Ph #e Val Ala Thr Thr Ala    -30               #   -25               #   -20Thr Ala Ala Ile Val Ala Ser Ala Val Ala Pr #o Val Val Ser Ala Ala-15                 - #10                   #-5              -1    #1Ser Phe Thr Asp Val Ala Pro Gln Tyr Lys As #p Ala Ile Asp Phe Leu              5     #              10     #              15Val Ser Thr Gly Ala Thr Lys Gly Lys Thr Gl #u Thr Lys Phe Gly Val         20          #         25          #         30Tyr Asp Glu Ile Thr Arg Leu Asp Ala Ala Va #l Ile Leu Ala Arg Val     35              #     40              #     45Leu Lys Leu Asp Val Asp Asn Ala Lys Asp Al #a Gly Phe Thr Asp Val 50                  # 55                  # 60                  # 65Pro Lys Asp Arg Ala Lys Tyr Val Asn Ala Le #u Val Glu Ala Gly Val                 70  #                 75  #                 80Leu Asn Gly Lys Ala Pro Gly Lys Phe Gly Al #a Tyr Asp Pro Leu Thr             85      #             90      #             95Arg Val Glu Met Ala Lys Ile Ile Ala Asn Ar #g Tyr Lys Leu Lys Ala        100           #       105           #       110Asp Asp Val Lys Leu Pro Phe Thr Asp Val As #n Asp Thr Trp Ala Pro    115               #   120               #   125Tyr Val Lys Ala Leu Tyr Lys Tyr Glu Val Th #r Lys Arg Leu Lys His130                 1 #35                 1 #40                 1 #45Gln Gln Ala Ser Val His Thr Lys Asn Ile Th #r Leu Arg Asp Phe Ala                150   #               155   #               160Gln Phe Val Tyr Arg Ala Val Asn Ile Asn Al #a Val Pro Glu Ile Val            165       #           170       #           175Glu Val Thr Ala Val Asn Ser Thr Thr Val Ly #s Val Thr Phe Asn Thr        180           #       185           #       190Gln Ile Ala Asp Val Asp Phe Thr Asn Phe Al #a Ile Asp Asn Gly Leu    195               #   200               #   205Thr Val Thr Lys Ala Thr Leu Ser Arg Asp Ly #s Lys Ser Val Glu Val210                 2 #15                 2 #20                 2 #25Val Val Asn Lys Pro Phe Thr Arg Asn Gln Gl #u Tyr Thr Ile Thr Ala                230   #               235   #               240Thr Gly Ile Lys Asn Leu Lys Gly Glu Thr Al #a Lys Glu Leu Thr Gly            245       #           250       #           255Lys Phe Val Trp Ser Val Gln Asp Ala Val Th #r Val Ala Leu Asn Asn        260           #       265           #       270Ser Ser Leu Lys Val Gly Glu Glu Ser Gly Le #u Thr Val Lys Asp Gln    275               #   280               #   285Asp Gly Lys Asp Val Val Gly Ala Lys Val Gl #u Leu Thr Ser Ser Asn290                 2 #95                 3 #00                 3 #05Thr Asn Ile Val Val Val Ser Ser Gly Glu Va #l Ser Val Ser Ala Ala                310   #               315   #               320Lys Val Thr Ala Val Lys Pro Gly Thr Ala As #p Val Thr Ala Lys Val            325       #           330       #           335Thr Leu Pro Asp Gly Val Val Leu Thr Asn Th #r Phe Lys Val Thr Val        340           #       345           #       350Thr Glu Val Pro Val Gln Val Gln Asn Gln Gl #y Phe Thr Leu Val Asp    355               #   360               #   365Asn Leu Ser Asn Ala Pro Gln Asn Thr Val Al #a Phe Asn Lys Ala Glu370                 3 #75                 3 #80                 3 #85Lys Val Thr Ser Met Phe Ala Gly Glu Thr Ly #s Thr Val Ala Met Tyr                390   #               395   #               400Asp Thr Lys Asn Gly Asp Pro Glu Thr Lys Pr #o Val Asp Phe Lys Asp            405       #           410       #           415Ala Thr Val Arg Ser Leu Asn Pro Ile Ile Al #a Thr Ala Ala Ile Asn        420           #       425           #       430Gly Ser Glu Leu Leu Val Thr Ala Asn Ala Gl #y Gln Ser Gly Lys Ala    435               #   440               #   445Ser Phe Glu Val Thr Leu Lys Asp Asn Thr Ly #s Arg Thr Phe Thr Val450                 4 #55                 4 #60                 4 #65Asp Val Lys Lys Asp Pro Val Leu Gln Asp Il #e Lys Val Asp Ala Thr                470   #               475   #               480Ser Val Lys Leu Ser Asp Glu Ala Val Gly Gl #y Gly Glu Val Glu Gly            485       #           490       #           495Val Asn Gln Lys Thr Ile Lys Val Ser Ala Va #l Asp Gln Tyr Gly Lys        500           #       505           #       510Glu Ile Lys Phe Gly Thr Lys Gly Lys Val Th #r Val Thr Thr Asn Thr    515               #   520               #   525Glu Gly Leu Val Ile Lys Asn Val Asn Ser As #p Asn Thr Ile Asp Phe530                 5 #35                 5 #40                 5 #45Asp Ser Gly Asn Ser Ala Thr Asp Gln Phe Va #l Val Val Ala Thr Lys                550   #               555   #               560Asp Lys Ile Val Asn Gly Lys Val Glu Val Ly #s Tyr Phe Lys Asn Ala            565       #           570       #           575Ser Asp Thr Thr Pro Thr Ser Thr Lys Thr Il #e Thr Val Asn Val Val        580           #       585           #       590Asn Val Lys Ala Asp Ala Thr Pro Val Gly Le #u Asp Ile Val Ala Pro    595               #   600               #   605Ser Lys Ile Asp Val Asn Ala Pro Asn Thr Al #a Ser Thr Ala Asp Val610                 6 #15                 6 #20                 6 #25Asp Phe Ile Asn Phe Glu Ser Val Glu Ile Ty #r Thr Leu Asp Ser Asn                630   #               635   #               640Gly Arg Arg Gln Lys Lys Val Thr Pro Thr Al #a Thr Thr Leu Val Gly            645       #           650       #           655Thr Lys Lys Lys Lys Lys Val Asn Gly Asn Va #l Leu Gln Phe Lys Gly        660           #       665           #       670Asn Glu Glu Leu Thr Leu Ser Thr Ser Ser Se #r Thr Gly Asn Val Asp    675               #   680               #   685Gly Thr Ala Glu Gly Met Thr Lys Arg Ile Pr #o Gly Lys Tyr Ile Asn690                 6 #95                 7 #00                 7 #05Ser Ala Ser Val Pro Ala Ser Ala Thr Val Al #a Thr Ser Pro Val Thr                710   #               715   #               720Val Lys Leu Asn Ser Ser Asp Asn Asp Leu Th #r Phe Glu Glu Leu Ile            725       #           730       #           735Phe Gly Val Ile Asp Pro Thr Gln Leu Val Ly #s Asp Glu Asp Ile Asn        740           #       745           #       750Glu Phe Ile Ala Val Ser Lys Ala Ala Lys As #n Asp Gly Tyr Leu Tyr    755               #   760               #   765Asn Lys Pro Leu Val Thr Val Lys Asp Ala Se #r Gly Lys Val Ile Pro770                 7 #75                 7 #80                 7 #85Thr Gly Ala Asn Val Tyr Gly Leu Asn His As #p Ala Thr Asn Gly Asn                790   #               795   #               800Ile Trp Phe Asp Glu Glu Gln Ala Gly Leu Al #a Lys Lys Phe Ser Asp            805       #           810       #           815Val His Phe Asp Val Asp Phe Ser Leu Thr As #n Val Val Lys Thr Gly        820           #       825           #       830Ser Gly Thr Val Ser Ser Ser Pro Ser Leu Se #r Asp Ala Ile Gln Leu    835               #   840               #   845Thr Asn Ser Gly Asp Ala Val Ser Phe Thr Le #u Val Ile Lys Ser Ile850                 8 #55                 8 #60                 8 #65Tyr Val Lys Gly Ala Asp Lys Asp Asp Asn As #n Leu Leu Ala Ala Pro                870   #               875   #               880Val Ser Val Asn Val Thr Val Thr Lys             885      #           890 <210> SEQ ID NO 7 <211> LENGTH: 498 <212> TYPE: DNA<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: streptavidin      gene <400> SEQUENCE: 7cccatggacc cgtccaagga ctccaaagct caggtttctg cagccgaagc tg#gtatcact     60ggcacctggt ataaccaact ggggtcgact ttcattgtga ccgctggtgc gg#acggagct    120ctgactggca cctacgaatc tgcggttggt aacgcagaat cccgctacgt ac#tgactggc    180cgttatgact ctgcacctgc caccgatggc tctggtaccg ctctgggctg ga#ctgtggct    240tggaaaaaca actatcgtaa tgcgcacagc gccactacgt ggtctggcca at#acgttggc    300ggtgctgagg ctcgtatcaa cactcagtgg ctgttaacat ccggcactac cg#aagcgaat    360gcatggaaat cgacactagt aggtcatgac acctttacca aagttaagcc tt#ctgctgct    420agcattgatg ctgccaagaa agcaggcgta aacaacggta accctctaga cg#ctgttcag    480 caataataag gatccggg              #                  #                   # 498 <210> SEQ ID NO 8 <211> LENGTH: 29<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      primer <400> SEQUENCE: 8 ttcatcgtaa acgccgaatt ttgtttctg         #                   #            29 <210> SEQ ID NO 9 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      primer <400> SEQUENCE: 9 agggaaatat atcaactctg caagtg          #                   #              26 <210> SEQ ID NO 10<211> LENGTH: 49 <212> TYPE: DNA<213> ORGANISM: Bacillus stearothermophilus <400> SEQUENCE: 10gaattcatcg atgtcgacca aggaggtcta gatggatccg gccaagctt  #               49

What is claimed is:
 1. A process for production of a crystalline S-layerprotein comprising: (a) transforming a gram-negative prokaryotic hostcell with a full length nucleic acid encoding an S-layer proteinselected from the group consisting of (i) a nucleic acid comprising anucleotide sequence from position 1 to 3684 of SEQ ID NO:1, (ii) anucleic acid comprising a nucleotide sequence which encodes an aminoacid sequence according to SEQ ID NO:2, and (iii) a nucleic acidcomprising a nucleotide sequence which hybridizes with at least one ofthe nucleic acid of (i) or (ii) under stringent conditions; (b)culturing the host cell under conditions which induce expression of thenucleic acid and production of the corresponding protein, and (c)isolating the protein from the host cell.
 2. The process as claimed inclaim 1, wherein the gram-negative prokaryotic host cell is an E. colihost cell.
 3. The process as claimed in claim 1, comprising isolatingthe protein from the interior of the host cell in the form of anassembled S-layer structure.
 4. The process as claimed in claim 1,wherein the nucleic acid encoding the S-layer protein comprises at leastone insertion encoding peptide or polypeptide sequences.
 5. The processas claimed in claim 4, wherein the at least one insertion is anucleotide sequence encoding a member selected from the group consistingof cysteine residues, regions with several charged amino acids ortyrosine residues, DNA-binding epitopes, metal-binding epitopes,immunogenic epitopes, allergenic epitopes, antigenic epitopes,streptavidin, enzymes, cytokines, and antibody-binding proteins.
 6. Theprocess as claimed in claim 5, wherein the at least one insertionencodes streptavidin.
 7. The process as claimed in claim 5, wherein theat least one insertion encodes immunogenic epitopes from a herpes virus.8. The process as claimed in claim 5, wherein the at least one insertionencodes enzymes comprising polyhydroxybutyric acid synthase or bacterialluciferase.
 9. The process as claimed in claim 5, wherein the at leastone insertion encodes cytokines comprising interleukins, interferons ortumour necrosis factors.
 10. The process as claimed in claim 5, whereinthe at least one insertion encodes antibody-binding proteins comprisingprotein A or protein G.
 11. The process as claimed in claim 5, whereinthe at least one insertion encodes antigenic epitopes which bindcytokines or endotoxins.
 12. The process as claimed in claim 5, whereinthe at least one insertion encodes metal-binding epitopes.
 13. Theprocess as claimed in claim 1, wherein a nucleic acid encoding agram-positive signal peptide is arranged in operative linkage at the 5′side of the nucleic acid encoding the S-layer protein.
 14. The processas claimed in claim 13, wherein the nucleic acid encoding the signalpeptide comprises (a) a signal peptide coding region of the nucleotidesequence of SEQ ID NO:1, (b) a nucleotide sequence which encodes anamino acid sequence according to SEQ ID NO:2, or (c) a nucleotidesequence that is at least 80% homologous to at least one nucleotidesequence of (a) or (b).
 15. An isolated nucleic acid encoding afull-length, crystalline recombinant S-layer protein selected from thegroup consisting of (i) a nucleic acid comprising a nucleotide sequencefrom position 1 to 3684 of SEQ ID NO:1, (ii) a nucleic acid comprising anucleotide sequence which encodes an amino acid sequence according toSEQ ID NO:2, and (iii) a nucleic acid comprising a nucleotide sequencewhich hybridizes with at least one of the nucleic acid of (i) or (ii)under stringent conditions, wherein the nucleic acid contains at leastone peptide or polypeptide-coding insertion within the region encodingthe S-layer protein, wherein the insertion is a site located at position582, 878, 917, 2504 or 2649 of the nucleotide sequence of SEQ ID NO. 1.16. A vector comprising at least one copy of a nucleic acid as claimedin claim
 15. 17. A transformed cell comprising a nucleic acid as claimedin claim 15 or a vector as claimed in claim 16, wherein the cell is agram-negative prokaryotic cell.
 18. A cell as claimed in claim 17,comprising a recombinant S-layer structure.
 19. A transformed cellwherein the cell is transformed with a nucleic acid as claimed in claim15.
 20. A transformed cell wherein the cell is transformed with a vectoras claimed in claim
 16. 21. The process according to claim 1, whereinthe nucleic acid of (i) does not contain a signal peptide-coding region.22. The process according to claim 13 wherein said stringent conditionsare washing at 55° C. in an aqueous low salt buffer comprising 0.2 ×SSC.23. The process according to claim 22, wherein said stringent conditionsare washing at 60° C. in an aqueous low salt buffer comprising 0.2 ×SSC.24. The nucleic acid according to claim 15, wherein the nucleic acid of(i) does not contain a signal peptide-coding region.
 25. The nucleicacid according to claim 15, wherein said stringent conditions arewashing at 55° C. in an aqueous low salt buffer comprising 0.2 ×SSC. 26.The nucleic acid according to claim 25, wherein said stringentconditions are washing at 60° C. in an aqueous low salt buffercomprising 0.2 ×SSC.
 27. The cell as claimed in claim 17, wherein thecell is E. coli in origin.
 28. The process as claimed in claim 7,wherein the herpes virus comprises herpes virus 6 or FMDV.
 29. A processfor production of a crystalline S-layer protein comprising a)transforming a gram-negative prokaryotic host cell with a full-lengthnucleic acid encoding an S-layer protein which comprises at least oneinsertion encoding peptide or polypeptide sequences and selected fromthe group consisting of (i) a nucleic acid comprising a nucleotidesequence from position 1 to 3684 of SEQ ID NO.1, (ii) a nucleic acidcomprising a nucleotide sequence which encodes the same amino acidsequence as the nucleic acid of (i), and (iii) a nucleic acid comprisinga nucleotide sequence which hybridizes with at least one of the nucleicacids of (i) or (ii) under stringent conditions; (b) culturing the hostcell under conditions which induce expression of the nucleic acid andproduction of the corresponding protein, and (c) isolating the proteinfrom the host cell.